Substrates and optical systems having a waveguide, nanometer-scale apertures, a lens array, and sensing regions and methods of use thereof

ABSTRACT

This invention provides substrates for use in various applications, including single-molecule analytical reactions. Methods for propagating optical energy within a substrate are provided. Devices comprising waveguide substrates and dielectric omnidirectional reflectors are provided. Waveguide substrates with improved uniformity of optical energy intensity across one or more waveguides and enhanced waveguide illumination efficiency within an analytic detection region of the arrays are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/949,313, filed Nov. 23, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/902,113, filed May 24, 2013, now U.S. Pat. No.9,222,123, which is a continuation of U.S. patent application Ser. No.13/274,547, filed Oct. 17, 2011, now U.S. Pat. No. 8,274,040, which is acontinuation of U.S. patent application Ser. No. 12/560,308, filed Sep.15, 2009, now U.S. Pat. No. 8,471,230, which claims the benefit of U.S.Provisional Application No. 61/192,326, filed Sep. 16, 2008, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

BACKGROUND OF THE INVENTION

A number of analytical operations benefit from the illumination ofsubstrates in order to accomplish the desired analysis. For example,interrogation of biopolymer array substrates typically employs wide areaillumination, e.g., in a linearized beam, flood or reciprocating spotoperation. Such illumination allows interrogation of larger numbers ofanalytical features, e.g., molecule groups, in order to analyze theinteraction of such molecule groups with a sample applied to the array.

For certain analytical operations, a tightly controlled illuminationstrategy is desirable. For example, it may be desirable to providestrict control of the volume of material that is illuminated, as well asthe overall area that is illuminated, effectively controllingillumination not only in the x or y axes of a planar substrate, but alsoin the z axis, e.g., extending away from the substrate. One example ofcontrolled illumination that accomplishes both lateral (x and y) andvolume (z) control is the use of zero-mode waveguides as a basesubstrate for analyzing materials. See, U.S. Pat. Nos. 6,991,726 and7,013,054, the full disclosures of which are incorporated herein byreference in their entireties for all purposes. Briefly, zero-modewaveguide array substrates employ an opaque mask layer, e.g., aluminum,chromium, or the like, deposited over a transparent substrate layer,through which are disposed a series of apertures through to thetransparent layer. Because the apertures are of sufficiently small crosssectional dimensions, e.g., on the order of 50-200 nm in cross section,they prevent propagation of light through them that is below a cut-offfrequency. While some light will enter the aperture or core, itsintensity decays exponentially as a function of the distance from theaperture's opening. As a result, a very small volume of the core isactually illuminated with a relevant level of light. Such ZMW arrayshave been illuminated using a number of the methods, including spotillumination, flood illumination and line illumination (using alinearized beam) (See, e.g., co-pending Published U.S. PatentApplication No. 2007-0188750, and published International PatentApplication No. WO 2007/095119, the full disclosures of which areincorporated herein by reference in their entireties for all purposes).

A second optical confinement strategy employs substrates that includewaveguides, such that the exponential decay of light outside thewaveguide may be exploited in a surface region of the substrate toselectively illuminate materials provided upon that surface.Waveguide-based illumination strategies can be used to illuminatematerials within ZMWs and other structures, such as wells positioned onthe surface. Further details regarding some such illumination schemescan be found in U.S. Patent Publication No. 2008-0128627, the fulldisclosure of which is incorporated herein by reference in its entiretyfor all purposes.

The present invention provides new substrates for waveguide arrays andmethods of illuminating analytes disposed upon the substrates, as willbe apparent upon review of the following.

SUMMARY OF THE INVENTION

Improved substrates of the invention find use in various applications,e.g., for analyte analysis, monitoring of enzymatic reactions, such asnucleic acid and polypeptide polymerization reactions, detecting bindingand other intermolecular interactions, genotyping, and many others. Thesubstrates, methods, and systems are particularly suitable fordetecting, monitoring, and analyzing single molecules, molecularcomplexes, or intermolecular reactions. As opposed to technologies thatallow only bulk detection, the ability to detect individual moleculesand reactions facilitates analyses that are not possible with bulkdetection, e.g., measurements of kinetics of an individual enzyme orenzyme complex in real time. For example, single polymerase or ribosomecomplexes can be monitored during polymerization of nucleic acids orpolypeptides, respectively.

The present invention provides substrates that include features, e.g.,waveguides, optical gratings, various conformations of surface regionsto be illuminated, various waveguide configurations, separate substratesfor optical splitting and biosensing functions, single substrates thatperform both optical splitting and biosensing functions, additionalsubstrate layers to reduce optical scattering and/or increase detectionefficiency, and substrates with improved analyte immobilizationcapabilities, which can be particularly desirable for theseapplications. The invention provides devices and methods that utilizeoptical gratings for normalizing optical energy intensity among arrayedwaveguides and enhancing waveguide-mediated illumination (or “waveguideillumination) efficiency within an analyte region of a substrate. Theinvention also provides waveguide configurations, e.g., waveguides at afirst depth within a substrate for distributing optical energy aroundthe substrate, and waveguides at a second depth disposed upon or withinthe substrate for illuminating analyte regions, to reduce propagationlosses within waveguides of a waveguide array. The invention alsoprovides tapered and multi-polarized waveguides, waveguides with taperedwaveguide cladding layers, waveguides with refractive indices that varyalong their length (e.g., in the core and/or cladding layer), andarrayed waveguides that are created from a single waveguide that passesback and forth within the waveguide substrate. The invention furtherprovides waveguides configured with a bend to reduce background signaland, thereby, increase the signal-to-background ratio. Further, theinvention provides waveguide arrays for delivering optical energy to aplurality of apertures, e.g., nanoholes or zero-mode waveguides. Theinvention also provides waveguide arrays that include an additionalsubstrate layer and or “dummy nanoholes” to reduce scattering effectsthat result from a plurality of analyte regions being disposed proximalto a waveguide core, as well as non-uniform spacing of the analyteregions disposed proximal to a waveguide core for enhanced propagationof optical energy through the core. The invention further provideswaveguide arrays that include deposition patterns of immobilizationcompounds for immobilizing analytes upon a substrate of the array. Inaddition, the invention provides waveguide substrates that allow biasedsurface chemistry within an aperture disposed upon the substrate, e.g.,such as the biased surface chemistry used in convention zero-modewaveguide applications. The invention also provides integrated opticalsystems that include microlens arrays, sensor arrays, multilayerdielectric stacks, and/or objective lenses positioned on both sides of awaveguide substrate.

In one aspect, the invention provides analytic devices for normalizingoptical energy intensity among arrayed waveguides comprising a substratecomprising a first surface, two or more waveguides disposed upon orwithin the substrate that are configured to receive optical energy at aportion of the two or more waveguides comprising an optical grating, andan analyte region disposed sufficiently proximal to a core of the atleast one of the two or more waveguides, such that the analyte region isilluminated by an evanescent field emanating from the core when opticalenergy is passed through the waveguide. The device can optionallycomprise a source of a single beam of optical energy, a diffractiveoptical element for splitting the single beam of optical energy into twoor more beams of optical energy, and/or a relay lens and microscopeobjective for focusing one of the two or more beams of optical energy atthe optical grating disposed within the waveguide. Optionally, theanalyte region of the devices is disposed within an aperture or welldisposed over an exposed surface of the waveguide, e.g., such as ananohole or a zero-mode waveguide. Optionally, the analyte region isdisposed within a nanometer-scale aperture that extends into thewaveguide. Further, the analyte region of the device can optionallycomprise an analyte.

In a related aspect, the invention provides methods for illuminating ananalyte comprising transmitting optical energy to two or more waveguidesdisposed upon or within a substrate and configured to receive opticalenergy, disposing an optical grating within the two or more waveguides,and disposing an analyte region sufficiently proximal to a core of atleast one of the two or more waveguides, such that the analyte isilluminated by an evanescent field emanating from the waveguide whenoptical energy is passed through the waveguide. Optionally, the methodcomprises providing a source of a single beam of optical energy,providing a diffractive optical element for splitting the beam into twoor more beams, and/or providing at least one relay lens and at least onemicroscope objective for focusing one of the two or more beams at theoptical grating disposed within the two or more waveguides.

The invention also provides analytic devices comprising a substratecomprising a first surface, at least one waveguide disposed upon withinthe substrate that comprises a diffraction grating pair, where thediffraction grating pair flanks a portion of the waveguide that isproximal to a detection region of the substrate and where thediffraction grating pair intensifies or reinforces optical energy of atleast one desired wavelength within the portion of the waveguide. Thedevice further comprises an analyte region disposed sufficientlyproximal to the detection region of the substrate and a core of thewaveguide, such that the analyte region is illuminated by an evanescentfield emanating from the waveguide core when optical energy is passedthrough the waveguide. Optionally, the analyte region of the device islocated within an aperture or well disposed over an exposed surface ofthe waveguide, e.g., such as a nanohole or a zero-mode waveguide. Incertain embodiments, the analyte region is disposed within ananometer-scale aperture that extends into the core of the waveguide.Further, the analyte region optionally comprises an analyte.

In a related aspect, the invention provides methods for illuminating ananalyte comprising illuminating at least one waveguide comprising adiffraction grating pair flanking a portion of the waveguide that isproximal to a detection region of the substrate, where the diffractiongrating pair intensifies or reinforces optical energy of at least onedesired wavelength within the portion of the waveguide, and disposing ananalyte sufficiently proximal to the detection region of the substrateand a core of the waveguide, such that the analyte is illuminated by anevanescent field emanating from the waveguide core when optical energyis passed through the waveguide. The analyte of the methods isoptionally disposed within an aperture or well disposed over an exposedsurface of the waveguide, e.g., such as a nanohole or a zero-modewaveguide. In certain embodiments, the analyte region is disposed withina nanometer-scale aperture that extends into the core of the waveguide.

The invention further provides analytic devices comprising a substratecomprising a first surface, at least one shallow waveguide disposed at afirst depth within the substrate, at least one deep waveguide disposedat a second depth within the substrate such that the shallow waveguideis disposed between the first surface and the deep waveguide, whereinthe shallow waveguide is optically coupled to the deep waveguide, and ananalyte region disposed sufficiently proximal to the shallow waveguide,such that the analyte region is illuminated by an evanescent fieldemanating from the core of the shallow waveguide when optical energy ispassed through the shallow waveguide. Optionally, the deep waveguide isshaped to enhance optical coupling between the deep waveguide and theshallow waveguide. Optionally, the analyte region of the devices isdisposed within an aperture or well disposed over an exposed surface ofthe waveguide, e.g., such as a nanohole or a zero-mode waveguide. Incertain embodiments, the analyte region is disposed within ananometer-scale aperture that extends into the core of the waveguide.Further, the analyte region of the devices can optionally comprise ananalyte.

In a related aspect, the invention provides methods for illuminating ananalyte comprising illuminating a deep waveguide disposed within asubstrate, coupling optical energy between the deep waveguide and ashallow waveguide disposed between a first surface of the substrate andthe deep waveguide, and disposing an analyte sufficiently proximal tothe shallow waveguide, such that the analyte is illuminated by anevanescent field emanating from the core of the shallow waveguide whenoptical energy is passed through the shallow waveguide. The analyte ofthe methods is optionally disposed within an aperture or well disposedover an exposed surface of the waveguide, e.g., such as a nanohole orspecific type of nanohole, e.g., a zero-mode waveguide. In certainembodiments, the analyte region is disposed within a nanometer-scaleaperture that extends into the core of the waveguide.

The invention provides analytic devices comprising at least onewaveguide disposed upon or within a substrate, wherein the waveguideterminates at a metal island that is penetrated by at least onenanometer-scale aperture, e.g., a zero-mode waveguide. The devices canoptionally comprise a plurality of waveguides terminating at a pluralityof metal islands, wherein each metal island is penetrated by at leastone nanometer-scale aperture. Optionally, the at least one waveguide ofthe devices is optically coupled to a plurality of secondary waveguidesthat collectively terminate at a plurality of metal islands, whereineach metal island is penetrated by at least one nanometer-scaleaperture. Optionally, secondary waveguides of the devices can beoptically coupled to a plurality of tertiary waveguides thatcollectively terminate at a plurality of metal islands, wherein eachmetal island is penetrated by at least one nanometer-scale aperture. Themetal islands of the devices optionally comprise a metal selected fromAl, Au, Ag, Ti, Pl, and Cr. Further, the metal islands that comprise atleast one nanometer-scale aperture, e.g., zero-mode waveguide, canoptionally comprise an analyte disposed within the aperture. The atleast one nanometer-scale aperture that comprises an analyte isoptionally disposed sufficiently proximal to the waveguide core, suchthat the analyte is illuminated by an evanescent field emanating fromthe waveguide core when optical energy is passed through the waveguide.In certain embodiments, the nanometer-scale aperture that extends intothe core of the waveguide.

In a related aspect, the invention provides methods for illuminating ananalyte comprising illuminating at least nanometer-scale aperture, e.g.,one zero-mode waveguide (ZMW), disposed in a metal island by providingoptical energy to the nanometer-scale aperture through an opticalwaveguide that terminates at a position proximal to the nanometer-scaleaperture, wherein the analyte is disposed within the nanometer-scaleaperture and illuminated by the optical energy emanating from a core ofthe optical waveguide and through the nanometer-scale aperture.Illuminating at least one nanometer-scale aperture optionally comprisescoupling optical energy from an originating waveguide to a plurality ofsecondary waveguides, where the optical waveguide that illuminates thenanometer-scale aperture is a secondary waveguide. Illuminating aplurality of nanometer-scale apertures optionally comprises couplingoptical energy from an originating waveguide to a plurality of secondarywaveguides that collectively terminate in a plurality of metal islandsthat comprise the plurality of nanometer-scale apertures. Illuminatingat least one nanometer-scale aperture optionally comprises couplingoptical energy from an originating waveguide to a plurality of secondarywaveguides, and coupling optical energy from the plurality of secondarywaveguides to a plurality of tertiary waveguides, where the opticalwaveguide that illuminates the nanometer-scale aperture is a tertiarywaveguide. Optionally, illuminating at least one nanometer-scaleaperture comprises coupling optical energy from the plurality ofsecondary waveguides to a plurality of tertiary waveguides thatcollectively terminate in a plurality of metal islands that comprise theplurality of nanometer-scale apertures.

The invention also provides analytic devices comprising a firstsubstrate that comprises an originating waveguide disposed upon orwithin the first substrate and two or more branch waveguides disposedupon or within the first substrate that are optically coupled to theoriginating waveguide. The devices further comprise a second substratecomprising two or more waveguides disposed upon or within the secondsubstrate, such that the two or more waveguides of the second substratehave a first end configured to be optically coupled to the two or morebranch waveguides of the first substrate. The second substrate alsocomprises an analyte region disposed sufficiently proximal to a core ofone of the two or more waveguides of the second substrate, such that theanalyte region is illuminated by an evanescent field emanating from thecore when optical energy is passed through the waveguides disposed uponor within the first substrate. Optionally, the cross-sectional area atthe first end of the two or more waveguides of the second substrate isgreater at the optical coupling location than the cross-sectional areaof the two or more waveguides of the second substrate at a detectionregion of the two or more waveguides of the second substrate. Thedevices optionally comprise a coupling element that couples opticalenergy between the first substrate and the second substrate. Thecoupling element optionally comprises at least one lens that focusesoptical energy from the two or more branch waveguides of the firstsubstrate toward the two or more waveguides of the second substrate.Optionally, the analyte region comprises an analyte. The secondsubstrate optionally comprises at least one aperture or well disposedover an exposed surface of the waveguide, e.g., such as a nanohole orspecific type of nanohole, e.g., a zero-mode waveguide, disposedproximal to at least one of the two or more waveguides within adetection region of the second substrate. In certain embodiments, thetwo or more branch waveguides disposed upon or within the firstsubstrate have tapered waveguide cores. In certain embodiments, theanalyte region is disposed within a nanometer-scale aperture thatpenetrates the first substrate in a region proximal to the core, andoptionally where the nanometer-scale aperture extends into the core.

In a related aspect, the invention provides methods for illuminating ananalyte comprising illuminating one or more distributing waveguidesdisposed upon or within a first substrate and coupling optical energyfrom the distributing waveguides to one or more receiving waveguides ofa second substrate, such that the analyte is sufficiently proximal to acore of at least one of the receiving waveguides of the second substrateto be illuminated by an evanescent field emanating from the core.Coupling optical energy optionally comprises focusing optical energyfrom the distributing waveguides of the first substrate through a lensto the one or more receiving waveguides of the second substrate. Incertain embodiments, the distributing waveguides have tapered waveguidecores.

Further, the invention provides analytic devices comprising at least afirst optical waveguide disposed within a substrate or upon or proximalto a first surface of the substrate, a mask layer disposed over a firstsurface of the substrate such that the mask layer covers at least aportion of the waveguide on the first surface and not covering at leasta second portion of the waveguide on the first surface, a mask claddinglayer disposed over the mask layer, and an analyte region disposedsufficiently proximal to a core of the first optical waveguide to beilluminated by an evanescent field emanating from the core when opticalenergy is passed through the first optical waveguide. The mask layeroptionally comprises a plurality of apertures that provide access to atleast a portion of the waveguide on the first surface. Optionally, thespacing between the apertures exhibits a random spacing error, e.g., arandom spacing error of about 5%. The mask cladding layer is optionallydisposed over the mask layer at locations where the mask layer isdisposed over the waveguide and not disposed over the mask layer atlocations where the mask layer is not disposed over the waveguide. Themask cladding layer optionally comprises a light reflective material,e.g., a metal (e.g., aluminum). Optionally, the mask cladding layercomprises a light absorptive material, e.g., Cr. The analyte regionoptionally is disposed within a nanometer-scale aperture or welldisposed over an exposed surface of the waveguide, e.g., such as ananohole (e.g., a zero-mode waveguide). Optionally, the analyte regionis disposed within a nanometer-scale aperture that extends into thefirst optical waveguide. Optionally, the analyte region comprises ananalyte.

In a related aspect, the invention provides methods for illuminating ananalyte disposed in an analyte region comprising distributing opticalenergy to the analyte region through an optical waveguide, such that theefficiency of optical energy delivery through the waveguide is enhancedby at least partially covering at least one surface of the waveguidewith an at least partially light reflective or light absorptive materialbilayer.

The invention also provides analytic devices comprising a substratecomprising a first surface, at least one optical waveguide disposed uponor within the first surface, an array of substantially parallel lines ofa surface immobilization compound such that the substantially parallelline of the surface immobilization compound are substantiallyperpendicular with respect to the at least one optical waveguide, and ananalyte attached to the surface immobilization compound where thesurface immobilization compound and the waveguide intersect, such thatthe analyte is disposed sufficiently proximal to a core of the opticalwaveguide to be illuminated by an evanescent field emanating from thecore when optical energy is passed through the optical waveguide.Optionally, the substrate comprises an array of optical waveguides. Thesurface immobilization compound optionally comprises a metal, e.g., Au.

In a related aspect, the invention provides methods for immobilizing ananalyte on an analytic device, comprising depositing an array ofsubstantially parallel lines of a surface immobilization compound on asubstrate of the analytic device, such that the substantially parallellines of the surface immobilization compound are deposited in asubstantially perpendicular orientation with respect to at least oneoptical waveguide disposed upon or within the substrate. The methodsfurther comprise attaching an analyte to the surface immobilizationcompound where the surface immobilization compound and the waveguideintersect. Optionally, the surface immobilization compound comprises ametal, e.g., Au. The substrate optionally comprises an array of opticalwaveguides.

The invention also provides analytic devices comprising a substratecomprising a detection region and at least one optical waveguide thattraverses the detection region, wherein the at least one opticalwaveguide has a first end coupled to an optical energy source and asecond end that is not coupled to the optical energy source, and furtherwherein the optical waveguide is configured to have a higher confinementof optical energy at the second end than at the first end. The devicefurther comprises a plurality of analyte regions disposed on a surfaceof the substrate in the detection region and sufficiently proximal to acore of the optical waveguide to be illuminated by an evanescent fieldemanating from the core when optical energy is passed through theoptical waveguide. In certain embodiments, the core of the opticalwaveguide is tapered such that there is a gradual decrease in thicknessfrom the first end to the second end. In certain embodiments, awaveguide cladding of the optical waveguide is tapered such that thecore becomes gradually closer to the analyte regions from the first endto the second end. In certain embodiments, the core has a firstrefractive index at the first end and a second refractive index at thesecond end, and further wherein the core is configured that there is agradual increase in refractive index from the first end to the secondend. In yet further embodiments, a waveguide cladding of the opticalwaveguide has a first refractive index at the first end and a secondrefractive index at the second end, and further wherein the core isconfigured that there is a gradual decrease in refractive index from thefirst end to the second end.

The invention also provides analytic devices comprising substrates withat least one optical waveguide, wherein the at least one opticalwaveguide is configured to propagate optical energy of a plurality ofwavelengths with comparable electric field intensities. The devices alsocomprise a plurality of analyte regions disposed on a surface of thesubstrate sufficiently proximal to a core of the optical waveguide to beilluminated by an evanescent field emanating from the core when opticalenergy is passed through the optical waveguide. In certain embodiments,the plurality of wavelengths are in the visible spectrum. In certainembodiments, the optical waveguide utilizes different polarizations foreach of the plurality of wavelengths, and, optionally, a first of thepolarizations utilizes a TE polarized mode, and a second of thepolarizations utilizes a TM polarized mode.

The invention also provides methods for providing uniform illuminationto a plurality of analyte regions on a substrate. For example, in someembodiments a waveguide core disposed within a substrate is configuredto gradually increase a measure of optical confinement of the waveguidecore along the direction of propagation of optical energy within thecore, e.g., in order to maintain a desired mode shape and or a desiredfield strength. A plurality of analyte regions are disposed along aportion of the substrate proximal to the waveguide core and opticalenergy is coupled into the waveguide core, thereby providing uniformillumination to the plurality of analyte regions. In certain preferredembodiments, the waveguide core is tapered so that it becomes thinneralong the direction of propagation of the optical energy in the core.Additionally or alternatively, the refractive index of the waveguidecore is gradually increased in the direction of propagation of theexcitation illumination. Additionally or alternatively, the waveguidecore has different polarizations for different wavelengths of excitationillumination.

The invention also provides an analytical device comprising a substrate,a detection region thereon, and at least one optical waveguide that isdisposed proximal to the detection region, wherein the detection regioncomprises a plurality of nanoholes within which analyte regions aredisposed, and a plurality of dummy nanoholes that do not compriseanalyte regions.

The invention also provides an analytical device that includes asubstrate comprising at least one optical waveguide and at least onenanometer-scale aperture that penetrates into a first side of thesubstrate and extends toward a core of the optical waveguide such thatan analyte disposed therein is sufficiently proximal to the core to beilluminated by an evanescent field emanating from the core when opticalenergy is passed through the optical waveguide. Optionally, the devicefurther comprises a detector disposed proximal to the substrate on aside opposite the first side, and a reflective coating over the firstside of the substrate that reflects signal emissions from thenanometer-scale aperture. The reflection of signal emissions by thereflective coating mitigates their passage through the first surface ofthe substrate and reflects them toward the detector. In certainembodiments, the reflective coating comprises aluminum. In certainembodiments, the nanometer-scale aperture penetrates a waveguidecladding and/or core of the optical waveguide. Optionally, the devicesfurther comprise a first objective lens positioned proximal to the firstside of the substrate, and a second objective lens positioned proximalto the substrate on a side opposite the first side. In certainembodiments, the substrate has a detection region that comprises thenanometer-scale aperture, and further wherein both the first and secondobjective lenses collect signal from the same portion or all of thedetection region. In certain embodiments, the substrate has a detectionregion that comprises the nanometer-scale aperture, and further whereinthe first objective lens collects signal from a first portion (e.g., afirst half) of the detection region and the second objective lenscollects signal from a second portion (e.g., a second half) of thedetection region. Optionally, the first and/or second objective lensesare water immersion lenses. In certain embodiments, the devices furthercomprise at least one detector operably linked to the first and thesecond objective lenses. Optionally, a first detector can be operablylinked to the first objective lens and a second detector can be operablylinked to the second objective lens; at least one of the first andsecond detectors can be a camera.

The invention also provides an analytical device that includes asubstrate comprising at least one optical waveguide having a core of ahigh refractive index material, a mask layer disposed over a firstsurface of the substrate, a thin layer disposed between the firstsurface of the substrate and the mask layer, wherein the thin layer issilane chemistry compatible, and one or more nanometer-scale apertures(e.g., zero-mode waveguides) disposed through the mask layer but notthrough the thin layer or into the substrate, wherein analyte regionswithin the apertures are sufficiently proximal to the core to beilluminated by an evanescent field emanating from the core when opticalenergy is passed through the optical waveguide. Optionally, the masklayer can comprise Al₂O₃ or a low refractive index material that iscoated with Al₂O₃.

The invention also provides an analytic device comprising a singlesubstrate that includes a coupling region in which optical energy iscoupled into an originating waveguide disposed upon or within thesubstrate; a splitter region in which the originating waveguide is splitinto two or more branch waveguides disposed upon or within thesubstrate, wherein the branch waveguides are optically coupled to theoriginating waveguide and therefore propagate the optical energy in theoriginating waveguide; a bend region wherein the two or more branchwaveguides comprise a bend that changes a direction of propagation ofthe optical energy within the branch waveguides; and a detection regionwherein at least one analyte region is disposed sufficiently proximal toat least one core of the branch waveguides to be illuminated by anevanescent field emanating from the core when optical energy is passedthrough the branch waveguides disposed upon or within the firstsubstrate. Optionally, the analytic device has a bend with an angle from45 to 135 degrees, preferably between about 75-105 degrees, and incertain embodiments 90 degrees. In certain embodiments, the analyteregion is disposed within a nanometer-scale aperture. In certainembodiments, the nanometer-scale aperture extends into a cladding layerproximal to the core, or extends all the way into the core. In certainembodiments, the nanometer-scale aperture is a zero-mode waveguide. Inpreferred embodiments, the splitter region comprises one or moreY-branch splitters and/or extends between about 10 mm and 50 mm.Optionally, the detection region of the device comprises a plurality ofoptically resolvable analyte regions, e.g., at least about 1000, or atleast about 10,000, or at least about 50,000 optically resolvableanalyte regions. In specific embodiments, a portion of one of the branchwaveguides that passes through the detection region is less than 3-5 mmin length.

The invention also provides an integrated optical device that comprisesa plurality of components, including a) a first component comprising awaveguide disposed upon or within a substrate, and further comprising abiosensing region wherein a plurality of nanometer-scale aperturescomprise analyte regions disposed sufficiently proximal to a core of thewaveguide to be illuminated by an evanescent field emanating from thecore when optical energy is passed through the waveguide; b) a secondcomponent comprising a microlens array that collects optical energysignals from the biosensing region and directs the optical energysignals so collected to a detector; and c) a third component comprisingthe detector. The first component optionally comprises a plurality ofchannel waveguides. The nanometer-scale apertures are optionallyzero-mode waveguides. In certain embodiments, the nanometer-scaleapertures extend into a cladding layer and/or the core of the waveguide.In certain embodiments, optical energy of at least twodetectably-different wavelengths is propagated within the waveguide.Optionally, each waveguide can use a different polarization mode topropagate each of the detectably-different wavelengths of opticalenergy. The second component optionally comprises a notch filter and/ora dispersive grating. In certain embodiments, the device furthercomprises an immersion fluid layer between the first and secondcomponents. The third component optionally comprises a multi-sensorarray and/or individual pixels that collect optical energy signals froma single one of the nanometer-scale apertures.

The invention also provides a device comprising a substrate comprisingone or more reaction sites, a mask layer on a first surface of thesubstrate, a multilayer dielectric stack, e.g., a dielectricomnidirectional reflector, on a second surface of the substrate, and anoptical energy source that directs optical energy into the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides an electric field distribution simulation for oneembodiment of a surface-exposed waveguide, and FIG. 1B provides aschematic representation of the electric field distribution simulation.

FIG. 2A provides an electric field distribution simulation for oneembodiment of a core-exposed waveguide, and FIG. 2B provides a schematicrepresentation of the electric field distribution simulation.

FIG. 3A provides an electric field distribution simulation for anembodiment of a core-exposed waveguide for which the shape and/orrefractive index of the waveguide core and substrate has been optimizedto concentrate the peak intensity of the electric field at the center ofa nanohole, and FIG. 3B provides a schematic representation of theelectric field distribution simulation.

FIGS. 4A, 4B, and 4C provide schematic illustrations of alternateexamples of the devices of the invention.

FIG. 5A schematically illustrates a cross-sectional view of an exemplarydevice of the invention that runs lengthwise through a channelwaveguide. FIG. 5B illustrates a cross-sectional view of the exemplarydevice depicted in FIG. 5A that runs perpendicular and through threearrayed channel waveguides.

FIG. 6 provides an illustrative example of a waveguide-illuminated ZMWarray comprising a nonmetal mask layer.

FIG. 7 schematically illustrates an analytic device that utilizeswaveguides of different depths within a substrate to distribute opticalenergy around the substrate.

FIG. 8 schematically illustrates a device for distributing opticalenergy to zero-mode waveguides disposed upon or within a substrate.

FIG. 9A schematically illustrates an overview of an analytic device thatincludes optical gratings for normalizing optical energy intensity amongtwo or more waveguides. FIG. 9B schematically illustrates a close-upview of an analytic device, which includes an optical grating etchedinto the cladding of a waveguide core, being illuminated by free spaceoptical energy.

FIG. 10 schematically illustrates an analytic device that includesoptical grating pairs for enhancing the illumination efficiency ofwaveguides within a detection region of the device.

FIG. 11 schematically illustrates an example waveguide array in whichthe ends of the arrayed waveguides are connected.

FIG. 12 provides an exemplary embodiment of a waveguide array thatperforms both optical splitting and biosensing functions.

FIG. 13 schematically illustrates an analytic device that utilizesseparate substrates for the optical splitting and biosensing functionsof the device.

FIG. 14 provides an exemplary embodiment of a waveguide array comprisingtapered waveguide cores in a splitter region of the substrate.

FIG. 15 schematically illustrates a device that utilizes a mask claddinglayer to reduce scattering of optical energy beyond a top surface of thedevice.

FIG. 16 illustrates a cross-section of an exemplary waveguide substratecomprising a metal layer disposed on a top surface of a waveguidecladding layer.

FIG. 17 provides a longitudinal phase matching diagram illustrating acorrugated waveguide output coupler.

FIG. 18 schematically illustrates a device for immobilizing a pluralityof analytes proximal to waveguides disposed upon or within a substrateof the device.

FIG. 19 provides one example of a system for use in the presentinvention that includes an optical train and detection system.

FIG. 20 provides an illustrative example of a device comprising awaveguide substrate and two objective lenses flanking a single detectionregion.

FIG. 21 provides an illustrative example of a device comprising awaveguide substrate and two objective lenses, each of which collectssignals from a discrete detection region.

FIG. 22 schematically illustrates an embodiment of an integrated opticaldevice comprising a waveguide substrate, a plurality of integratedmicroarrays, and a detector.

FIG. 23 schematically illustrates an embodiment of an integrated opticaldevice comprising a waveguide substrate, a plurality of integratedmicroarrays, and a sensor array.

FIG. 24 schematically illustrates an embodiment of a device of theinvention comprising a dielectric omnidirectional reflector.

DETAILED DESCRIPTION

The present invention is broadly applicable to any application in whichone desires to illuminate materials (e.g., analytes) that are at orproximal to a surface and/or specific locations on a surface, withoutilluminating materials that are not similarly situated. For example,such systems are particularly useful in the analysis of individualmolecules or molecular interactions and/or interactions ofsurface-coupled reactants, such as, e.g., polynucleotide or polypeptidepolymerization reactions, hybridization reactions, binding assays, andthe like. Further details regarding such single-molecule/molecularcomplex analyses are provided, e.g., in U.S. patent application Ser. No.12/413,258 (filed Mar. 27, 2009), Ser. No. 12/328,715 (filed Dec. 4,2009), Ser. No. 12/413,226 (filed Mar. 27, 2009), and 61/186,661 (filedJun. 12, 2009); U.S. Patent Publication No. 20070206187; and U.S. Pat.Nos. 7,056,661 and 6,917,726, all of which are incorporated herein byreference in their entireties for all purposes.

In certain embodiments, the present invention provides substratescomprising one or more waveguides. The methods includewaveguide-mediated illumination of an analytical reaction or analyte ofinterest using, e.g., surface-exposed, substrate-enclosed, orcore-exposed waveguides, such that the exponential decay of lightoutside the waveguide core (e.g., an evanescent field) may be exploitedon a surface region of the substrate to illuminate materials providedupon that surface. By providing materials at or proximal to the surface,e.g., at a reaction site, one can controllably illuminate such materialswithout illuminating any materials outside of the evanescent field. Incertain embodiments, the present invention provides devices comprisingmultilayer dielectric stacks, e.g. dielectric omnidirectionalreflectors, configured to propagate optical energy to one or morereaction sites on a substrate.

A number of analytical operations can benefit from the ability tocontrollably illuminate materials at or near a surface and/or at anumber of locations thereon, without excessively illuminating thesurrounding environment. Examples of such analyses include illumination,observation and/or analysis of surface-bound cells, proteins, nucleicacids, or other molecules of interest. Such illumination is particularlyuseful in illuminating fluorescent and/or fluorogenic materials upon orproximal to the surface, including nucleic acid array-based methods,substrate-coupled nucleic acid and polypeptide sequencing-by-synthesismethods, antibody/antigen interactions, binding assays, and a variety ofother applications. The methods, devices, compositions, and systemsprovided herein are particularly suitable for single-molecule-leveldetection of analytical reactions in real-time, e.g., during the ongoinganalytical reaction. For example, a single polymerase enzyme can beimmobilized on a substrate and monitored as it incorporatesdifferentially labeled nucleotides into a nascent nucleic acid strand byilluminating the substrate surface where the polymerase is bound anddetecting a sequence of fluorescent emissions from the reaction sitethat corresponds to the sequence of nucleotides incorporated by thepolymerase.

In the context of analysis, the substrates and methods of the inventionare advantageous for numerous reasons. For example, because theillumination light is applied in a spatially focused manner, e.g.,confined in at least one lateral and one orthogonal dimension, usingefficient optical systems, e.g., fiber optics, waveguides, multilayerdielectric stacks (e.g., dielectric reflectors), etc., the inventionprovides an efficient use of illumination (e.g., laser) power. Forexample, illumination of a substrate comprising many separate reactionsites, “detection regions,” or “observation regions” using waveguidearrays as described herein can reduce the illumination power ˜10- to1000-fold as compared to illumination of the same substrate using a freespace illumination scheme comprising, for example, separate illumination(e.g., via laser beams) of each reaction site. In general, the higherthe multiplex (i.e., the more surface regions to be illuminated on thesubstrate), the greater the potential energy savings offered by thewaveguide and dielectric stack-based illumination schemes providedherein. In addition, since waveguide illumination need not pass througha free space optical train prior to reaching the surface region to beilluminated (as described further below), the illumination power can befurther reduced.

In addition, because illumination is provided from within confinedregions of the substrate itself (e.g., optical waveguides), issues ofillumination of background or non-relevant regions, e.g., illuminationof non-relevant materials in solutions, autofluorescence of substrates,and/or other materials, reflection of illumination radiation, etc. aresubstantially reduced. Likewise, this aspect of the invention providesan ability to perform many homogenous assays for which it would begenerally applicable.

In addition to mitigating autofluorescence of the substrate materials,the systems described herein substantially mitigate autofluorescenceassociated with the optical train. In particular, in typicalfluorescence spectroscopy, excitation light is directed at a reaction ofinterest through at least a portion of the same optical train used tocollect signal fluorescence, e.g., the objective and other optical traincomponents. As such, autofluorescence of such components will contributeto the detected fluorescence level and can provide signal noise in theoverall detection. Because the systems provided herein direct excitationlight into the substrate through a different path, e.g., through anoptical fiber optically coupled to the waveguide in the substrate, or byinternal reflection between a mask layer and a dielectric reflector,this source of autofluorescence is eliminated.

Waveguide-mediated and dielectric-based illumination is alsoadvantageous with respect to alignment of illumination light withsurface regions to be illuminated. In particular, substrate-basedanalytical systems, and particularly those that rely upon fluorescent orfluorogenic signals for the monitoring of reactions, typically employillumination schemes whereby each analyte region must be illuminated byoptical energy of an appropriate wavelength, e.g., excitationillumination. While bathing or flooding the substrate with excitationillumination serves to illuminate large numbers of discrete regions,such illumination suffers from the myriad complications described above.To address those issues, some embodiments of the invention providetargeted excitation illumination to selectively direct separate beams ofexcitation illumination to individual reaction regions or groups ofreaction regions, e.g. using waveguide arrays. When a plurality, e.g.,hundreds or thousands, of analyte regions are disposed upon a substrate,alignment of a separate illumination beam with each analyte regionbecomes technically more challenging and the risk of misalignment of thebeams and analyte regions increases. In the present invention, alignmentof the illumination sources and analyte regions is built into thesystem, because the illumination pattern and reaction regions areintegrated into the same component of the system, e.g., a waveguidesubstrate. In certain preferred embodiments, optical waveguides arefabricated into a substrate at defined regions of the substrate, andanalyte regions are disposed upon the area(s) of the substrate occupiedby the waveguides.

Finally, the substrates of the invention typically are provided fromrugged materials, e.g., silicon, glass, quartz or polymeric or inorganicmaterials that have demonstrated longevity in harsh environments, e.g.,extremes of cold, heat, chemical compositions, e.g., high salt, acidicor basic environments, vacuum and zero gravity. As such, they providerugged capabilities for a wide range of applications.

Exemplary waveguide array configurations, methods of fabricating thewaveguide arrays of the invention, waveguide arrays with additionalfunctionalities, devices comprising multilayer dielectric stacks (e.g.,dielectric reflectors), and methods and applications provided by thepresent invention are described in detail below.

I. Waveguide Substrates

Waveguide substrates of the present invention generally include amatrix, e.g., a silica-based matrix, such as silicon, glass, quartz orthe like, polymeric matrix, ceramic matrix, or other solid organic orinorganic material conventionally employed in the fabrication ofwaveguide substrates, and one or more waveguides disposed upon or withinthe matrix, where the waveguides are configured to be optically coupledto an optical energy source, e.g., a laser. Such waveguides may be invarious conformations, including but not limited to planar waveguidesand channel waveguides. Some preferred embodiments of waveguidesubstrates comprise an array of two or more waveguides, e.g., discretechannel waveguides, and such waveguide substrates are also referred toherein as waveguide arrays. Further, channel waveguides can havedifferent cross-sectional dimensions and shapes, e.g., rectangular,circular, oval, lobed, and the like; and in certain embodiments,different conformations of waveguides, e.g., channel and/or planar, canbe present in a single waveguide substrate.

In typical embodiments, a waveguide comprises a waveguide core and awaveguide cladding adjacent to the waveguide core, where the waveguidecore has a refractive index sufficiently higher than the refractiveindex of the waveguide cladding to promote containment and propagationof optical energy through the core. In general, the waveguide claddingrefers to a portion of the substrate that is adjacent to and partially,substantially, or completely surrounds the waveguide core, as furtherdescribed below. The waveguide cladding layer can extend throughout thematrix, or the matrix may comprise further “non-cladding” layers. A“substrate-enclosed” waveguide or region thereof is entirely surroundedby a non-cladding layer of matrix; a “surface-exposed” waveguide orregion thereof has at least a portion of the waveguide cladding exposedon a surface of the substrate; and a “core-exposed” waveguide or regionthereof has at least a portion of the core exposed on a surface of thesubstrate. Further, a waveguide array can comprise discrete waveguidesin various conformations, including but not limited to, parallel,perpendicular, convergent, divergent, entirely separate, branched,end-joined, serpentine, and combinations thereof.

A surface or surface region of a waveguide substrate is generally aportion of the substrate in contact with the space surrounding thesubstrate, and such space may be fluid-filled, e.g., an analyticalreaction mixture containing various reaction components. In certainpreferred embodiments, substrate surfaces are provided in apertures thatdescend into the substrate, and optionally into the waveguide claddingand/or the waveguide core. In certain preferred embodiments, suchapertures are very small, e.g., having dimensions on the micrometer ornanometer scale, and are further described below.

It is an object of the invention to illuminate analytes (e.g., reactioncomponents) of interest and to detect signal emitted from such analytes,e.g., by excitation and emission from a fluorescent label on theanalyte. Of particular interest is the ability to monitor singleanalytical reactions in real time during the course of the reaction,e.g., a single enzyme or enzyme complex catalyzing a reaction ofinterest. The waveguides provided herein provide illumination via anevanescent field produced by the escape of optical energy from thewaveguide core. The evanescent field is the optical energy field thatdecays exponentially as a function of distance from the waveguidesurface when optical energy passes through the waveguide. As such, inorder for an analyte of interest to be illuminated by the waveguide itmust be disposed near enough the waveguide core to be exposed to theevanescent field. In preferred embodiments, such analytes areimmobilized, directly or indirectly, on a surface of the waveguidesubstrate. For example, immobilization can take place on asurface-exposed waveguide, or within an aperture in the substrate. Insome preferred aspects, analyte regions are disposed in apertures thatextend through the substrate to bring the analyte regions closer to thewaveguide core. Such apertures may extend through a waveguide claddingsurrounding the waveguide core, or may extend into the core of thewaveguide. In certain embodiments, such apertures also extend through amask layer above the surface of the substrate. In preferred embodiments,such apertures are “nanoholes,” which are nanometer-scale holes or wellsthat provide structural confinement of analytic materials of interestwithin a nanometer-scale diameter, e.g., ˜10-100 nm. In someembodiments, such apertures comprise optical confinementcharacteristics, such as zero-mode waveguides, which are alsonanometer-scale apertures and are further described elsewhere herein.Although primarily described herein in terms of channel waveguides, suchapertures could also be constructed on a planar waveguide substrate,e.g., where the planar waveguide portion/layer is buried within thesubstrate, i.e., is not surface-exposed. Regions on the surface of awaveguide substrate that are used for illumination of analytes aregenerally termed “analyte regions,” “reaction regions,” or “reactionsites,” and are preferably located on a surface of the substrate nearenough to a waveguide core to be illuminated by an evanescent waveemanating from the waveguide core, e.g., on a surface-exposed waveguideor at the bottom of an aperture that extends into the substrate, e.g.,into the waveguide cladding or core. The three-dimensional area at areaction site that is illuminated by the evanescent field of a waveguidecore (e.g., to an extent capable of allowing detection of an analyte ofinterest) is generally termed the “observation volume” or “illuminationvolume.” A region of a waveguide substrate that comprises one or moreanalyte regions is generally referred to as a “detection region” of thesubstrate, and a single substrate may have one or multiple detectionregions.

An electric field distribution simulation for a surface-exposedwaveguide is shown in FIG. 1A; the simulation data was generated incolor, but is shown here in grayscale, which does not allow distinctionof various aspects of the distribution. As such, a schematicrepresentation of the electric field distribution simulation is shown inFIG. 1B to show the general pattern of the changing electric field inand around the waveguide core. Specifically, highest intensity portionof the field is black, with the color lightening as the field intensitydecreases. In such a surface-exposed waveguide, peak electric intensitygenerally lies along the center of the waveguide core. On the exposedsurface of the core, the electric field intensity is roughly 20% of theintensity at the center of the core, and this field intensity decaysexponentially into the space above the exposed surface of the core,e.g., into a fluid volume, thereby providing excitation confinement inthe vertical direction. In the horizontal direction, however, theconfinement is weak. That is, a relatively large area on the surface ofthe waveguide experiences a relatively strong field intensity. As such,the relatively large observation volume generated by the evanescentfield from the surface exposed waveguide core may be greater than apreferred observation volume, e.g., for single molecule detection. Forexample, even in an embodiment in which a confocal pinhole is used onthe surface of the waveguide, the observation volume is stillapproximately 500-fold larger than the observation volume of a typicalzero-mode waveguide. As such, higher background signal is expected underconditions in which concentrations of detectable reaction components,e.g. fluorescent-labeled reactants, are high enough that more than oneis expected to reside in a single such observation volume at a giventime.

FIG. 2A provides an illustrative example of an electric fielddistribution simulation for one example of a core-exposed waveguidecomprising a waveguide core into which a nanohole has been disposed. Asfor FIG. 1A, the simulation data was generated in color, but is shownhere in grayscale, which does not allow distinction of various aspectsof the distribution. As such, a schematic representation of the electricfield distribution simulation is shown in FIG. 2B to show the generalpattern of the changing electric field in and around the waveguide core.Specifically, highest intensity portion of the field is black, with thecolor lightening as the field intensity decreases. Such a structurephysically limits the volume of a solution or reaction mixture exposedto the electric field of the waveguide core, thereby limiting theobservation volume within which excitation of and emission from variousreaction components can occur. Therefore, for a given concentration oflabeled reactant in a reaction mixture, fewer individual labeledreactants would exist in the observation volume of a nanohole than wouldbe expected to exist in the observation volume on a surface-exposedwaveguide core, e.g., because the observation volume of the former ismuch smaller than the observation volume of the latter. Take, forexample, a waveguide core of 0.5 μm² that lies 150 nm below the surfaceof the substrate and comprises a nanohole with an 80 nm diameter thatextends from the surface of the substrate to the center of the waveguidecore. A simulated electric field based on a 633 nm wavelength ofexcitation light and water as the fluid in the nanohole produces theelectric field distribution shown in FIG. 2A. The presence of thenanohole in the core of the waveguide changes the electric fielddistribution, and focuses the highest intensity of excitation radiationat the base of the nanohole in the center of the waveguide. Further,modification of the shape and/or refractive index of the waveguide coreand substrate can alter the electric field to further concentrate thepeak intensity at the center of the nanohole, as shown in FIG. 3A andthe schematic representation depicting the general pattern of thechanging electric field provided in FIG. 3B, in which the most intenseportion of the electric field distribution is better centered in thebottom of the nanohole than the electric field distribution provided inFIGS. 2A and 2B. In general, the faster the intensity decays from thebottom of the nanohole, the better the illumination confinement withinthe nanohole. Although the nanohole itself does not provide confinementof the excitation radiation, the effective detection region is confinedin the nanometer range because labeled reactants can only get into theobservation volume via the nanohole. Thus, the observation volume of ananohole extending into the core of a waveguide can be comparable tothat of a zero-mode waveguide, e.g., at the attoliter (10⁻¹⁸ L) tozeptoliter (10⁻²¹ L) scale, a volume suitable for detection and analysisof single molecules and single molecular complexes.

In certain preferred embodiments, a mask layer is disposed upon thewaveguide substrate, and analyte regions are disposed through the masklayer such that materials within the analyte regions can be sufficientlyproximal to the waveguide core to permit their illumination by anevanescent field emanating from the waveguide core during operation ofthe array. The analyte regions are generally disposed through the masklayer within an area of the mask layer that demarks a detection regionof the waveguide substrate. When a waveguide substrate is employed aspart of a larger analytical system, e.g., a system for detectingfluorescent materials that are proximal to the waveguide surface, adetection system can be disposed proximal to the detection region of thearray such that signals derived from illuminated materials within theanalyte regions can be detected and subsequently analyzed.

The present invention provides waveguide substrates with improvedoptical and/or structural functionalities that provide improvedillumination energy distribution across arrays of reaction regions,improved illumination of individual reaction regions, and a number ofother improved properties. For example, in certain aspects, the presentinvention provides waveguides of different depths for enhanced opticalenergy distribution and illumination of analyte regions. Also providedare waveguide arrays that include grating couplers for normalizingoptical energy intensity among arrayed waveguides and/or grating pairsdisposed upon the surface of arrayed waveguides for enhancedillumination efficiency within a detection region of the array. Further,the present invention provides waveguide substrates with a top maskcladding layer disposed upon a mask layer for reduced scattering oflight resulting from nanoholes situated proximal to the waveguides, aswell as non-uniform spacing of such nanoholes for reduced backreflection of light into the waveguides. The present invention furtherprovides waveguide substrates with lines of surface immobilizationcompounds deposited upon the surface of the substrate for improvedimmobilization of analytes upon or proximal to one or more waveguidecores. The present invention further provides waveguide substrates withwaveguides having a tapered structure and/or a gradation of refractiveindex that is gradually modified in the direction of optical energypropagation to in order to smoothly adjust the degree of confinement andrelative field strength, as further described below.

Example Waveguide Configuration

An object of the instant invention is to provide confinement ofillumination from a waveguide core to analyte regions disposed on asurface of a waveguide substrate. In certain preferred embodiments, suchconfinement is facilitated by a high refractive index contrast betweenthe waveguide core and the waveguide cladding around the waveguide core.In certain preferred embodiments, a waveguide cladding surrounding awaveguide core has a significantly lower refractive index than thewaveguide core, and serves to confine the modal profile (or diameter) ofthe guided optical wave(s) into the submicrometer (or only a fewmicrometer) range. That is, the optical waves extend only asubmicrometer to few micrometer distance outside the core. At adetection region of the substrate, nanosize features (e.g., nanoholes orzero-mode waveguides) provide illumination confinement to excite singleanalytes for detection, e.g., by fluorescence emission. In certainpreferred embodiments, the optical waves are in the visible range. Incertain preferred embodiments, multiple wavelengths of optical waves arepropagated and detected simultaneously, e.g., in real time during thecourse of an analytical reaction of interest.

An example analytic device that employs arrayed waveguides to illuminatea plurality of analyte regions, e.g., optically confined regions inwhich materials of interest are controllably illuminated in the mannerdescribed above, is schematically illustrated in FIGS. 4A-C. FIG. 4Aschematically illustrates a top view of example device 400 that includesa matrix 402. Waveguides 406-416 are provided to confine and propagatelight introduced into them, For the purposes of the present disclosure,a waveguide refers to a waveguide core and can further include awaveguide cladding layer partially or substantially surrounding thecore. These exemplary arrayed waveguides receive light from anexcitation illumination source, e.g., via a first optical fiber 403,that is optically coupled to the waveguides, e.g., connected such thatlight is transmitted from one to the other, propagated (via waveguide404), and optionally divided among branch waveguides 406-416. It will beappreciated that the number of arrayed waveguides of the device canrange from one waveguide to a plurality of waveguides, e.g., 10 or more,20 or more, 30 or more, 40 or more, 100 or more, or 1000 or morewaveguides are possible. A mask layer (see, e.g., 420 in FIG. 4B) isprovided, such that analyte regions, e.g., apertures disposed throughthe mask layer (See, e.g., 422 in FIG. 4B), can be disposed over andprovide access to a portion of the surface of waveguides 406-416 atdiscrete locations within detection region 418 of device 400. While thesubstrates of the invention are preferably planar substrates having thewaveguide(s) disposed therein, it will be appreciated that for certainapplications, non-planar substrates may be employed, including, forexample, fiber based substrates, shaped substrates, and the like.Although example device 400 is shown with a single input waveguide 404,it will be appreciated that waveguide substrates of the invention maycomprise multiple input waveguides, and that these multiple inputwaveguides can be divided into branch waveguides that illuminate one ormore detection regions. For example, in certain embodiments a singledetection region is illuminated with optical energy originating frommultiple input waveguides, each of which is optionally split into aplurality of branch waveguides prior to passage through the detectionregion. Such multiple input waveguides may be coupled to one or moreoptical energy sources along a single edge of a waveguide substrate, ormay be coupled on different sides, e.g. opposite sides, of the waveguidesubstrate. The one or more optical energy sources may provide the sameor different optical energy to the multiple input waveguides, e.g., sameor different wavelengths, intensities, etc.

A cross section through the detection region of example device 400,where the section runs lengthwise and through an arrayed waveguide,e.g., waveguide 410, is schematically illustrated in FIG. 4B. As shown,mask layer 420 is disposed on top of waveguide 410, which includeswaveguide core 409 and waveguide cladding 411. Analyte regions areprovided as apertures through the mask layer that provide access to theunderlying surface-exposed waveguide. For example, as shown, nanohole422, is disposed through mask layer 420, thereby providing access to thesurface of waveguide 410, and in particular to the surface of thewaveguide cladding. Nanoholes, e.g., nanometer-sized apertures or wellsdisposed through mask layer 420, are preferable as compared to largerapertures, e.g., microholes, milliholes, centiholes, etc., becausenanoholes are of such small cross sectional dimensions, e.g., 50-200 nmin cross section, that they provide a sufficiently small volume ofstructural confinement such that issues of illumination of background ornon-relevant regions, e.g., illumination of non-relevant materials insolutions, are substantially reduced.

As optical energy is passed through waveguide 410 in the direction asindicated by arrow 421, a portion of the volume of nanohole 422 isilluminated by evanescent field 423, as the field extends into nanohole422. As a result, only those reactants that are disposed at or near theexposed surface of the waveguide, within the nanohole, are subjected tosufficient illumination intensity, e.g., to emit a fluorescent signal.In some cases, the structure of the mask layer 420 and aperturestherein, e.g., nanoholes 422, may provide optical confinement within theapertures to attenuate the illumination that enters the reaction region.For example, in some embodiments, apertures disposed within a mask layerare zero-mode waveguides.

The mask layer masks some portions of the waveguide but not otherportions, which remain accessible to materials disposed over the overallsubstrate. In particular, the evanescent wave from exposed waveguideregion can reach reagents deposited over the surface of the overallsubstrate, and particularly within analyte regions. By virtue of themask layer, the evanescent wave from the other blocked portions of thewaveguide will not reach any materials deposited over the surface of thesubstrate. As a result, one can pre-select those regions that areoptically interrogable, and thus direct optical systems appropriately.

A cross section through the detection region of example device 400,where the section runs perpendicular and through three arrayedwaveguides, e.g., waveguides 406, 408 and 410, is schematicallyillustrated in FIG. 4C. (Each waveguide has a separate waveguide coreand waveguide cladding, not shown.) Again, as shown, analyte regions,e.g., nanoholes 422, 424 and 426 are disposed through mask layer 420,exposing the surface of waveguides 406-410. A portion of nanoholes422-426 can be illuminated by an evanescent field (not shown) emanatingfrom waveguides 406-410 as light passes through the waveguides. Analyticprocesses occurring in nanoholes 422-426 may be observed by detectionsystem 428.

A further exemplary analytic device that employs arrayed waveguides toilluminate a plurality of analyte regions, e.g., optically confinedregions in which materials of interest are controllably illuminated inthe manner described above, is schematically illustrated in FIGS. 5A-B.FIG. 5A schematically illustrates a cross-sectional view of exampledevice 500, where the section runs lengthwise and through a channelwaveguide 510 comprising waveguide core 509 and waveguide cladding 511.Device 500 includes a matrix 502. The channel waveguide core 509 has ahigher refractive index than that of the waveguide cladding 511. A masklayer 520 is also illustrated on the surface of a top portion of thematrix 502. The analyte regions are disposed within apertures (e.g.,nanohole 522) that extend through the mask layer 520, the top portion ofthe matrix 502, and the waveguide cladding 511, and extend into thewaveguide core 509. As described above, nanoholes are preferable ascompared to larger apertures, e.g., microholes, milliholes, centiholes,etc., because nanoholes are of such small cross sectional dimensions,e.g., 50-200 nm in cross section, that they provide a sufficiently smallvolume (e.g., attoliter- to zeptoliter-scale) of structural confinementsuch that issues of illumination of background or non-relevant regions,e.g., illumination of non-relevant materials in solutions, aresubstantially reduced, which facilitates detection and interrogation ofsingle molecules or molecular complexes.

As optical energy is passed through waveguide 510 in the direction asindicated by arrow 521, a portion of the volume of nanohole 522 isilluminated by evanescent field 523, as the field passes throughnanohole 522. As a result, only those reactants that are disposed at ornear the evanescent field emanating from the waveguide, e.g., within anillumination volume within nanohole 522, are subjected to sufficientillumination, e.g., to emit a fluorescent signal. In some cases, thestructure of the mask layer 520 and apertures, e.g., nanohole 522, mayprovide optical confinement within the apertures to attenuate theillumination that enters the reaction region. For example, in someembodiments, apertures disposed within a mask layer are zero-modewaveguides. Although the evanescent field shown in FIG. 5A does notextend to the mask layer, one of ordinary skill will readily recognizethat different intensities of light can be propagated in channelwaveguide 510, so in other embodiments an evanescent wave may extend tothe mask layer. In such embodiments, the presence of an opaque masklayer can prevent excitation of reagents disposed over the surface ofthe substrate. Further, at least a portion of any signal emitted fromreagents outside of the apertures (e.g., excited by light passingthrough the top of the aperture 522) is blocked from re-entering theaperture, thereby reducing background noise.

A cross-section through a detection region of example device 500, wherethe section runs perpendicular and through three arrayed waveguides,e.g., waveguides 506-510, is schematically illustrated in FIG. 5B.Again, as shown, analyte regions, e.g., nanoholes 522-526 are disposedthrough mask layer 520, through the top portion of the matrix 502, andthrough the waveguide claddings into the waveguide cores of waveguides506, 508, and 510. A portion of nanoholes 522-526 can be illuminated byan evanescent field (not shown) emanating from waveguides 506-510 aslight passes through them. Analytic processes occurring in nanoholes522-526 may be observed by detection system 528.

The waveguide arrays of the present invention are in no way limited bythe example waveguide arrays described above and illustrated in FIGS.4A-C and 5A-B, as additional configurations and functionalities arepossible, including those described below.

Waveguide Array Fabrication

In some cases, the waveguides described herein are generally producedusing conventional ion implantation techniques to selectively ion dopeselected regions of substrates, e.g., SiO₂ based substrates, to providepatterned regions of higher refractive index, so as to function aswaveguides embedded in the underlying substrate. Examples of suchdevices are disclosed in, e.g., Marcuse, Theory of Dielectric OpticalWaveguides, Second Ed. (Academic Press 1991). Alternate waveguidefabrication processes and configurations are equally applicable to thepresent invention, including hybrid material waveguides, e.g., employingpolymeric materials as a portion or all of the subject substrate, e.g.,a polymer core having a first refractive index, disposed within asubstrate of another material having a second refractive index, whichmay be polymeric, or another material, e.g., silicon, glass, quartz,etc. For example, waveguides of the invention can be produced bydepositing Si₃N₄ via chemical vapor deposition (CVD), e.g., low pressurechemical vapor deposition (LPCVD).

Waveguide substrates including mask layers may be prepared by a varietyof known fabrication techniques. For example, lithographic techniquesmay be used to define the mask layer out of polymeric materials, such asphotoresists, using e.g., conventional photolithography, e-beamlithography, or the like. Alternatively, lithographic techniques may beapplied in conjunction with layer deposition methods to deposit metalmask layers, e.g., using aluminum, gold, platinum, chrome, or otherconventionally used metals, or other inorganic mask layers, e.g., silicabased substrates such as silicon, SiO₂, or the like. Alternatively,negative tone processes may be employed to define pillars of resiststhat correspond to the apertures, e.g., nanoholes (See, e.g., U.S. Pat.No. 7,170,050, previously incorporated herein by reference). The masklayer is then deposited over the resist pillars and the pillars aresubsequently removed. In particularly preferred aspects, both theunderlying substrate and the mask layer are fabricated from the samematerial, which in particularly preferred aspects, is a transparentsubstrate material such as an SiO₂ based substrate such as glass,quartz, or fused silica. By providing the mask and underlying layers ofthe same material, one can ensure that the two layers have the sameinteractivity with the environments to which they are exposed, and thusminimize any hybrid surface interactions.

In the case of SiO₂ based substrates and mask layers, conventionalfabrication processes may be employed. In particular, a glass substratebearing the surface exposed waveguide has a layer of resist depositedover its surface. A negative of the mask layer is then defined byappropriate exposure and development of the resist layer to provideresist islands where one wishes to retain access to the underlyingwaveguide. The mask layer is then deposited over the surface and theremaining resist islands are removed, e.g., through a lift off process,to provide the openings to the underlying waveguides. In the case ofmetal layers, deposition may be accomplished through a number of means,including evaporation, sputtering or the like. Such processes aredescribed in, e.g., U.S. Pat. No. 7,170,050, which is incorporatedherein by reference in its entirety for all purposes. In the case ofsilica based mask layers, a CVD process may be employed to deposit asilicon layer onto the surface. Following lift off of the resist layer,a thermal oxidation process can convert the mask layer to SiO₂.Alternatively, etching methods may be used to etch access points tounderlying waveguides using conventional processes. For example, asilicon layer may be deposited over an underlying waveguide substrate. Aresist layer is then deposited over the surface of the silicon layer andexposed and developed to define the pattern of the mask. The accesspoints are then etched from the silicon layer using an appropriatedifferential etch to remove silicon but not the underlying SiO₂substrate. Once the mask layer is defined, the silicon layer is againconverted to SiO₂ using, e.g., a thermal oxidation process.

In addition to the advantages of reduced autofluorescence, waveguidesubstrates having an integrated mask layer provide advantages of opticalalignment over similar arrays of wells or structures that areilluminated through non-integrated optical paths. For example, in somecases, illuminating an ordered array of reaction regions with minimalexcess illumination involves directing excitation illumination at thevarious regions by presenting a corresponding array of illuminationspots. In doing so, one must take substantial care in aligning theoptical presentation of the illumination spots to the ordered array ofreaction regions. Such alignment presents challenges of both design androbustness, as such systems may be prone to drifting or othermisalignment influences. Where, as in the present invention,illumination is integrated or “hard wired” into the substrate by virtueof the integrated waveguide, alignment is automatic as a result of thesubstrate fabrication process. Further the possibility of loss ofalignment over time, e.g., drift, is eliminated.

In other cases, surface features may include other confinementstrategies, including, e.g., chemical surface functionalities that areuseful in a variety of surface analytical operations, such ashydrophobic coatings or hydrophilic coatings that are optionallypatterned, to provide confinement or direction to aqueous materials,chemical derivatization, e.g., to facilitate coupling of otherfunctional groups or otherwise, e.g., by providing hydrophobic barrierspartially or completely surrounding a desired region, or by providingimmobilized coupling groups in desired reaction regions forimmobilization of specific reagents. As will be appreciated, in somecases, particularly where structural confinement is provided upon thesurface of the substrate, it may not be necessary to divide up lightthrough a series of discrete waveguides in a given substrate. Inparticular, because one can obtain a desired level of multiplex andspatial separation organization from structurally dividing up thesurface, one need not obtain that property through the use of separatewaveguides. In such cases, a single field waveguide disposed at thesurface of the substrate will suffice to deliver light to the variousreaction regions on the surface of the substrate, e.g., as defined bythe mask layer.

In addition to structures and strategies that provide for positioningand/or confinement upon a substrate surface, other components may beprovided upon a substrate, including backside coatings for thesubstrate, e.g., antireflective coatings, optical indicator components,e.g., structures, marks, etc. for the positioning and or alignment ofthe substrate, its constituent waveguides, and/or for alignment of othercomponents. Other components may include substrate packaging components,e.g., that provide fluidic interfaces with the substrate surface, suchas flow cells, wells or recesses, channel networks, or the like, asmacrostructures as compared to the surface defined structures above, aswell as alignment structures and casings that provide structuralprotection for the underlying substrates and interactive functionalitybetween the substrates and instrument systems that work with/analyze thesubstrates.

Waveguide Configurations and Structures

While primarily illustrated with respect to waveguide arrays thatinclude a plurality of parallel waveguides, the invention may alsoinclude patterned waveguides that have a variety of differentconfigurations, including serpentine waveguides, branched waveguides,interleaved waveguides, divergent waveguides, convergent waveguides orany of a variety of configurations depending upon the desiredapplication. For example, where it is desired to provide evanescentillumination to larger areas of the substrate, it may be desirable toprovide non-linear waveguides, such as serpentine waveguides, as well aslarger area waveguides, such as wider or slab waveguide(s), oralternatively and likely preferably, larger numbers of parallel orsimilarly situated waveguides. The waveguide substrates may include asingle waveguide that may span a fraction of the width of the substrateor substantially all of that width. In accordance with preferred aspectshowever, waveguide arrays are used to split individual originating beamsinto two or more waveguides, preferably more than 10 waveguides, morethan 20 waveguides, more than 40 waveguides, and in some cases more than50 waveguides or even more than 100, 1000, 5000 or more waveguides. Thenumber of waveguides may typically vary greatly depending upon the sizeof the substrate used, and the optical resolution of the detectionsystem, e.g., its ability to distinguish materials proximal to differentwaveguides.

The waveguides may individually vary in the size of the core region inorder to vary the evanescent field that one can access. Typically, thewaveguides will have a cross sectional dimension of from about 0.1 toabout 10 μm, preferably from about 0.2 to about 2 μm and more preferablyfrom about 0.3 to about 0.6 μm, and may be circular, oval, rectangular,lobed, or flattened (e.g., wide in the z dimension and narrow in the ydimension, or vice versa). In addition, the cross sectional dimension ofa waveguide may be continuous or vary along the length of the waveguide.A variety of other waveguide dimensions may be employed as well,depending upon the desired application. For example, in some cases, asingle waveguide may be used where the cross-sectional dimension of thewaveguide is substantially the same as the substrate width or length,e.g., a single waveguide that substantially spans a substrate's width.Notwithstanding the foregoing, preferred aspects will provide arrayedwaveguides, e.g., multiple waveguides typically arranged in parallellinear format.

A variety of different waveguide structures are exploitable in thepresent invention. In particular, the waveguide arrays of the inventionmay employ embedded and/or channel waveguides. Details regardingwaveguide structures that can be employed in the present invention areprovided in Lundquist et al., U.S. Patent Publication No. 2008-0128627,entitled SUBSTRATES, SYSTEMS AND METHODS FOR ANALYZING MATERIALS,incorporated herein by reference in its entirety for all purposes. Forpurposes of the invention, a waveguide that is referred to as beingdisposed upon or within the surface of a substrate encompasseswaveguides that are disposed on but above the surface, within thesubstrate but at or exposed to the surface, or are disposed within thesubstrate, but sufficiently proximal to the surface that the evanescentwave from light passing through the waveguide still reaches above thesurface to provide an illumination volume.

II. Waveguide Arrays with Additional Functionalities

The waveguide arrays of the present invention can include additionaloptical functionalities, including, e.g., specific types of nanoscaleapertures such as zero-mode waveguides (ZMWs) that provide opticalconfinement of illumination light in addition to structural confinement.Further details regarding ZMWs can be found in U.S. Pat. Nos. 6,991,726and 7,013,054, previously incorporated herein by reference. Otheroptical functionalities that may be integrated into or upon thesubstrates include lenses, filters, antireflective coatings, or thelike. Other functionalities may be incorporated into the fabricatedsubstrate that operate on and/or in conjunction with the waveguides orwaveguide arrays of the invention. For example, optical switching orattenuation components may be provided upon or within the substrates ofthe invention to selectively direct and/or modulate the light passingthrough a given waveguide or waveguides.

In addition to the optical functionalities of the substrates of theinvention, in some cases, such substrates may include additionalfunctionalities that provide a defined region on the substrate surfaceto limit the access that reagents or other elements have to theillumination zone above a waveguide. For example, in some cases, thesubstrates may include a patterned structure or set of structures overthe surface of the substrate providing selected exposure of the surfaceexposed waveguide(s). Such selected regions may provide limited areas ofillumination on a given substrate by blocking the illumination regionexisting above other portions of the waveguide(s), such as a mask layer.As a result, only selected portions of the surface will be within theillumination or detection region of the waveguides. Such regions may beselected to align with detection systems or the requirements of suchsystems, e.g., sample spacing permitting spectral separation of signalsfrom each region (See, e.g., U.S. patent application Ser. No.11/704,733, filed Feb. 9, 2007, which is incorporated herein byreference in its entirety for all purposes). In addition to limitedaccess, such structures may also provide structural confinement ofreactions or their components, such as wells or channels. In one aspect,for example, microfluidic channels may be provided disposed over surfaceexposed waveguide or waveguide array. Such channels may be independentlyused to deliver different reagents to different portions of a waveguideor waveguide array.

Waveguide Arrays for Enhanced Optical Energy Propagation

The present invention provides devices for waveguide-based illuminationof analyte regions in apertures (e.g., nanoholes or ZMWs) that reducethe variation in illumination over the length of the waveguide, forexample, by mitigating propagation losses over the length of thewaveguide. Such propagation losses can be further exacerbated by a metallayer disposed over the surface of the substrate, because it can absorboptical energy from a surface-exposed or core-exposed waveguide, or evena waveguide near to the surface of the substrate. Such metal layers aretypically found in conventional ZMW arrays, presenting a challenge forcombining such arrays with waveguide illumination strategies.

One of the limitations of waveguide illumination is optical attenuationas the light propagates down the guide resulting in a reduction in powerat different locations in the guide. For example, a particular laserintensity coupled into the waveguide will experience a slow decrease inenergy density as light travels down the guide due to propagationlosses, with the highest power at the end nearest the illuminationsource and the lowest at the end farthest from the illumination source.The degree of the propagation loss is typically a function of thedesigned geometry and manufacturing tolerances, and presents a challengeto performing multiplexed analytical reactions because it constrains thespatial range of the usable waveguide structure. It is important tomaximize the distance over which the laser intensity is sufficientlyuniform, in order to maximize the multiplex capabilities of the system.It is therefore an object of the present invention to provide uniformpower over the length of a waveguide, e.g., to promote uniformillumination of all reaction sites to be illuminated by the waveguide.

In certain embodiments, a waveguide is tapered such that the coregradually becomes thinner along the direction of propagation. Thiscauses the degree of light confinement to be gradually increased, whichcan offset the gradual reduction in the total amount of power in theguide due to propagation losses and essentially maintain a desired modeshape and field strength for the optical energy propagated over thelength of the waveguide core. In principle, the sum of propagationlosses is balanced by the decreasing core size such that uniformity ofevanescent field strength can be held constant for an arbitrarily longdistance, with limitations to the strength of the evanescent field alsobeing dependent on the starting laser power and the starting waveguidecore dimensions. For a given waveguide substrate, once the propagationloss is determined the waveguide geometry can be designed to smoothlyvary, thereby modifying the degree of confinement such that the relativefield strength at the point of interest increases at the same rate thatpropagation losses reduce the total power in the guide. For example, atapered waveguide core can be widest at the portion most proximal to thelight source, slowly narrowing along the guide, with the fieldlocalization increasing at the same rate that propagation losses arereducing the waveguide field strength. The tapering can take place inany direction including the z direction (top to bottom), the y direction(side to side), or a combination thereof.

In certain embodiments, a waveguide cladding above a waveguide core in awaveguide substrate is tapered such that the waveguide core is slowlybrought closer to the reaction sites at the surface of the substrate byan ever-decreasing width of the waveguide cladding layer that separatesthe core from the reaction sites. As such, although there is propagationloss from a waveguide with a non-tapered geometry, as the field strengthin the waveguide decreases, it is brought closer to the reaction sites,and this increasing proximity compensates for an overall reduced fieldstrength. In some embodiments, both the waveguide cladding layer andwaveguide core are be tapered to mitigate loss of field strength due topropagation losses.

In other embodiments, the refractive index of the waveguide core orwaveguide cladding layer is gradually modified along the propagationdirection to smoothly adjust the degree of confinement and relativefield strength. These waveguide substrates can be fabricated usingstandard waveguide manufacturing techniques known in the art. Further,embodiments of the invention may comprise combinations of variousstrategies for enhancing optical energy propagation and mitigatingpropagation loss, e.g., varying the taper, depth, and/or refractiveindices of waveguide cores and/or waveguide claddings.

In certain aspects, a waveguide designed to propagate light of multipledifferent wavelengths provides various benefits to methods, devices, andsystems of the invention. However, for a waveguide of a givengeometrical and material structure, different propagation wavelengthsbehave differently. For example, for available material structures thereis no solution that is single-mode at both 488 nm and 643 nm, andalthough a waveguide can be designed to be single-mode for twowavelengths, such a waveguide would not be expected to promote similarevanescent field strength for both wavelengths. Although similar fieldstrength may be achieved by increasing the laser power of the shorterwavelength laser, this could cause the total power required for thedevice to be several times larger and is undesirable for variousreasons, including higher autofluorescence, increased heat in thewaveguide (which could damage the substrate and/or increase thetemperature at a reaction site), and inefficient use of laser power. Amulti-mode structure would experience modal interference that wouldcause different locations along the guide to show very different fieldstrengths, and so also does not provide a practical means to propagatemultiple different wavelengths of light in a substantially uniformmanner over the length of a waveguide.

An object of the invention is to provide a desired evanescent fieldstrength for light of multiple wavelengths at a specific location or setof locations, e.g., reaction sites. A further object of the invention isto detect optical energy of multiple different wavelengths, e.g., in thevisible spectrum, simultaneously in real time, e.g., during an ongoingenzymatic reaction. In preferred embodiments, a waveguide designed toachieve the same or similar evanescent field strength for multiplewavelengths without the undesirable side-effects of using a single-modeor multi-mode waveguide is provided. The field profile of guided modesof different wavelengths are not equivalent, and in general the fartherapart the wavelengths, the larger the difference in field strength. Incertain preferred embodiments, the invention provides a “polarizedwaveguide” designed to utilize a different polarization for differentexcitation wavelengths to be simultaneously propagated in the waveguide.In planar waveguides, p-polarized modes are more tightly confined thans-polarized modes. In channel guides the propagation solutions arepreferably divided into two polarization categories, TE and TM polarizedmodes. In this way, a single waveguide geometry can be an adjustablesolution to propagation of light of two different wavelengths. Thedifferent polarizations for the different wavelengths can be designed toprovide the same evanescent field strength for both wavelengths at thedesired location(s). Polarization can be manipulated independently inseveral ways that are well known in the art. For example, a polarizingprism can be used to combine and perfectly overlap two different lightbeams. A wave retarding plate can also be used to independentlymanipulate the polarizations of two beams having different wavelengths.The Faraday effect can be used to independently manipulate two beamseven after they are collinear. Finally, diffraction gratings andHolographic Optica Elements (HOE's) can be used to direct multiple beamsof different wavelengths separately to achieve the desired alignment andefficient coupling into a planar waveguide structure with independentpolarization control. In certain embodiments, the waveguides arespecifically tapered to provide the same evanescent field strength anddesired mode shape for both wavelengths. In certain embodiments ofmulti-laser systems different polarizations can be assigned to bestbalance the field strength. In certain preferred embodiments, thewavelengths of light propagated in a polarized waveguide are in thevisible range (e.g., between about 380 nm and 750 nm; or in terms offrequency, between about 790 and 400 terahertz). In certain preferredembodiments, excitation light of at least two different wavelengths issimultaneously propagated in a single waveguide and used to illuminatedifferent fluorescently labeled analytes in an illumination volume at areaction site, emission light is emitted from the fluorescently labeledanalytes in response to the excitation illumination, and the emissionlight is detected in real time. In certain preferred embodiments, lightof at least two different wavelengths is propagated in a singlewaveguide, the wavelength of a first differs from the wavelength of asecond by at least 50 nm, but also desirable are 100 nm, 200 nm, 300 nm,and 400 nm separations. The desired separation of a particularconfiguration is determined by both the waveguide structure, thenanohole structure, and the chemical fluorophores involved in theapplication.

ZMW array substrates typically employ an opaque mask layer, e.g.,aluminum, chromium, or the like, deposited over a transparent substratelayer. A series of apertures are disposed through the mask layer to thetransparent layer. Disposing a plurality of ZMWs proximal to, and along,a core-exposed waveguide for waveguide-based illumination of ZMWsentails the waveguide being situated immediately beneath a metal layer.As will be appreciated, over the length of the waveguide, propagationlosses can occur due to the metal layer being in direct contact with thewaveguide core. Such propagation losses may present difficulties forutilizing the waveguide for both transporting optical energy around thesubstrate and illuminating the reaction regions, e.g., ZMWs, disposedproximal to the waveguide core.

In certain embodiments, such propagation losses can be mitigated bysubstituting the metal mask layer for a nonmetal mask layer. Anillustrative example of such a waveguide-illuminated ZMW is provided isFIG. 6 in which a waveguide core 609 made of a high refractive indexmaterial (e.g., LiNbO₃, n=2.3; Si₃N₄, n=2; SiO_(x), N_(y), n=1.55 to 2;etc.) is combined with a mask layer 620 made of aluminum oxide (Al₂O₃,n=1.7) and an intervening thin layer of glass 630 (e.g., fused silica).The mask layer 620 is patterned over the glass layer 630 to generate theZMW structure 622. This type of waveguide-illuminated ZMW providesbenefits beyond the mitigation of propagation losses, as well. Forexample, it provides optical confinement of the evanescent field to thereaction site in an observation volume similar to those of conventionalZMW arrays, at least in part due to shorter evanescent decay lengths,which results in lower background signal, shorter diffusive residencetimes, and an overall lower signal-to-noise ratio when monitoringanalytical reactions illuminated by the waveguide. Further, conventionalZMW surface chemistry can also be used in these waveguide-illuminatedZMW arrays because the surface properties are the same, e.g., allowingbiased immobilization of reaction components to the bottom, but not thesides of the ZMW (or vice versa). In general, the length of evanescentdecay within the ZMW becomes shorter and the optical confinement becomesbetter as the refractive index of the core is increased and/or as therefractive index difference between the core and the mask layer is high.In certain embodiments, the thin glass layer can be substituted forother materials that support biased surface chemistries within the ZMW,and preferably support conventional ZMW surface chemistry, which isdescribed elsewhere in the art (see below). In addition, the mask layercan be low refractive index materials other than aluminum oxide (suchas, e.g., CVD and PECVD silicon oxide, Spin-on-Glass orboro-phosphoslicate glass (BPSG), etc.) that is subsequently coated withaluminum oxide to facilitate biased surface chemistry. The propertiesand methods of use of such materials are known to those of ordinaryskill in the art. Of course, the glass floor of the ZMW would need to beprotected from the aluminum oxide during the coating process. Methodsfor conventional surface chemistry in ZMWs is provided, e.g., inKorlach, et al. (2008) Proc. Natl. Acad. Sci. 105(4):1176-1181; U.S.Patent Publication Nos. 20070077564, 20070134128, 20070238679,20080241892, 20080032301, 20080050747, and 20080220537; and U.S. patentapplication Ser. No. 11/645,125, filed Dec. 21, 2006, the disclosures ofall of which are incorporated herein by reference in their entiretiesfor all purposes.

In some exemplary embodiments, variations in (conveyed) illuminationover the distance of the waveguide may be achieved by providingfunction-specific waveguides within the substrate and coupling them. Inparticular, one may employ a first waveguide for the unimpeded transportof excitation illumination, which is then optically coupled to a secondwaveguide that serves to deliver the excitation illumination to thereaction region. In such cases, the devices provided by the inventionmay include both “shallow” and “deep” waveguides. The shallow waveguidese.g., waveguides disposed just beneath the metal ZMW layer near the topsurface of the substrate, function to illuminate the ZMWs within adetection region of the array. The deep waveguides are buried within thesubstrate at a distance further from the metal layer of ZMWs than theshallow waveguides, and function to transport power around the substratewithout the propagation losses associated with waveguides situated nearthe ZMW layer. Although described below primarily with reference to ZMWarrays, combinations of waveguides of differing depths can also be usedto illuminate reaction sites on other types of waveguide substrates,e.g. planar waveguide substrates or waveguide substrates comprisingapertures other than ZMWs, e.g., other types of nanometer-scaleapertures.

As will be appreciated, optical energy transported through deepwaveguides can be coupled to shallow waveguides by a variety of means.For example, an evanescent field emanating from the deep waveguides,e.g., a light field that decays exponentially as a function of distancefrom the deep waveguide surface, can be exploited to illuminate theshallow waveguides. Coupling optical energy between the deep and shallowwaveguides can be enhanced by altering the shape of the deep waveguide,e.g., tapering the cross sectional area of the deep waveguide such thatthe cross sectional area of the deep waveguide is smaller at regions ofthe array where coupling between the deep and shallow waveguide isdesired. For example, a smaller cross sectional area of the waveguide ata given position permits the evanescent field to extend a greaterdistance from the deep waveguide core at that position. For example, adecreased cross sectional area of the deep waveguide core can ensurethat the evanescent field extends to, or beyond, the portion of theshallow waveguide on the side opposite the deep waveguide, therebyproviding maximal illumination of the shallow waveguide by the deepwaveguide. In other embodiments, the matrix separating the deepwaveguide from the shallow waveguide is varied to allow more efficienttransfer of optical energy to the shallow waveguide in desired regions,e.g., detection regions. Coupling between the deep and shallowwaveguides can also be enhanced by altering the optical properties ofthe core or cladding, e.g., the thickness or index of refraction of thecladding, of the deep waveguides. Further, a “leaky-mode” in the deepwaveguide can be created by patterning and etching a shallow grating onit. The grating can be optimized, e.g., by altering the period, dutycycle and/or depth of the grating, to enhance optical coupling to theshallow waveguide. Other methods for coupling the deep and shallowwaveguides known to those of ordinary skill in the art are alsocontemplated.

A cross section of the detection region of an example device thatemploys shallow and deep waveguides to illuminate a plurality of ZMWs isschematically illustrated in FIG. 7. As shown, substrate 700 is providedincluding top metal layer 702 through which holes for the formation ofZMWs 704, 706 and 708 are etched. Shallow waveguide 710 lies justbeneath the ZMW layer. Deep waveguide 712 is disposed within substrate700 beneath shallow waveguide 710. Deep waveguides 712 transportsoptical energy around the device with mitigated propagation lossresulting from metal layer 702 at least in part due to its distance frommetal layer 702. As shown, optical energy is coupled from deepwaveguides 712 to shallow waveguides 710, permitting ZMW illumination byshallow waveguides 710 with enhanced efficiency as compared toillumination by a waveguide responsible for both transporting opticalenergy across the device and ZMW illumination.

In addition to the above-described waveguide arrays that addresspropagation losses associated with a metal ZMW layer disposed upon thewaveguides, the present invention also provides waveguide arrays inwhich individual waveguides terminate in metal islands that include oneor more ZMWs. By providing each metal island with its own illuminationguide entrance, the propagation losses associated with guidingillumination light beneath an extensive metal surface, e.g., acontinuous metal layer providing a plurality of ZMWs, are eliminated.The metal islands of the devices can comprise a broad variety of metalsknown to those of ordinary skill in the art and disclosed elsewhereherein, including but not limited to Al, Au, Ag, Pt, Ti, and Cr.

An example waveguide array in which metal islands including one or moreZMWs are illuminated by waveguides is schematically illustrated in FIG.8. As shown, primary waveguide 800 is disposed upon or within substrate802. Secondary waveguides 804 and 806 are configured to receive opticalenergy from primary waveguide 800. Tertiary waveguides 808, 810, 812 and814 are configured to receive optical energy from secondary waveguides804 and 806. As shown, tertiary waveguides 808-814 terminate in metalislands 816, 818, 820 and 822 that include a ZMW. It will be appreciatedthat the metal island can also include two or more ZMWs. The ZMWs can bedisposed through the metal islands such that the ZMW is aligned with,and disposed upon or proximal to, the external surface of tertiarywaveguides 808-814, permitting illumination of the ZMWs by an evanescentfield emanating from tertiary waveguides 808-814 as optical energypasses through the tertiary waveguides.

Waveguide Arrays for Improved Uniformity of Analyte Region Illumination

Conventional optical splitters, e.g., Y splitters or T splitters, areoften employed for splitting optical energy from an originatingwaveguide into 2 or more branch waveguides. To split optical energy to,e.g., 32 (or N) waveguides, a star coupler or tree coupler can be used.A tree coupler comprises multiple stages of 1×2 Y splitters, e.g., 5stages for a 1×32 splitter. For conventional Y splitters working attelecom wavelength (1550 nm), the splitting error is approximately 0.2dB (2.3%), i.e., the power difference between the two branches is about0.2 dB (2.3%). For a 5-stage 1×32 splitter, the compound splitting errorcan reach 1 dB (11%). For a splitter working at visible wavelength, theexpected splitting error would scale up with the frequency of thewavelength. Thus, the compound splitting error for a visible 1×32splitter could reach as high as 30-40%. This error can result ininconsistent illumination among arrayed waveguides and, accordingly,analyte regions disposed proximal to the waveguides. Non-uniform analyteregion illumination can adversely affect the functionality of ananalytic device, e.g., a waveguide array and optical detection systemfor illumination and observation of a plurality of molecular processes.The present invention provides waveguide arrays that exhibitsubstantially uniform optical energy intensity among the arrayedwaveguides.

The invention provides waveguide arrays for improved uniformity ofanalyte region illumination that include, e.g., optical gratingsdisposed upon an external surface of the waveguides. The gratings can beconfigured to couple free space light between a source of free spacelight and the waveguides. By providing each waveguide with gratings ofuniform characteristics, e.g., uniform grating period, cross sectionalarea of the slits that make up the grating, and the like, optical energyis coupled to the waveguide cores such that illumination of thewaveguides with optical energy of a desired intensity and wavelength isachieved. Because the gratings normalize the intensity of optical energyamong the arrayed waveguides, the waveguides produce substantiallyuniform evanescent fields that emanate from the waveguides as lightpasses through the waveguides. Accordingly, analyte regions disposedproximal to the arrayed waveguides are illuminated with improveduniformity, and issues involving analyte regions receiving too much ortoo little illumination light are substantially reduced.

An example device for achieving substantially uniform optical energyintensity among arrayed waveguides is schematically illustrated in FIG.9A. As shown, substrate 900 is provided including a number of opticalwaveguides, e.g., surface-exposed waveguides 902, 904 and 906. Opticalenergy source 914 is provided for illumination of waveguides 902-906.Optical energy source 914 provides a single beam of optical energy 916,e.g., a single laser beam, which directs optical energy towardreconfigurable diffractive optical element 918. Reconfigurablediffractive optical element 918 splits the single beam of optical energyinto multiple beams of optical energy, e.g., multiple laser beams 920,922 and 924. Multiple laser beams 920-924 are passed through relaylens/microscope objective 926 to generate parallel and focused beams ofoptical energy, e.g., parallel and focused laser beams 928, 930 and 932.As shown, parallel and focused laser beams 928-932 are directed towardoptical gratings, e.g., optical gratings 908, 910 and 912, disposedwithin a waveguide cladding layer (not shown) of, or proximal to,surface-exposed waveguides 902-906, respectively.

A more detailed schematic illustration of optical gratings for couplingoptical energy to a waveguide array is presented in FIG. 9B. Substrate934 is provided including optical waveguide 936 and waveguide claddinglayer 938 disposed proximal to optical waveguide core 936. Opticalgrating 940 can comprise submicrometer wide holes or slits periodicallyetched through waveguide cladding layer 938 proximal to waveguide core936. The angle of incidence 942 of focused beam of optical energy 944can be reconfigured and optimized according to the period of opticalgrating 940 to achieve optical energy of a desired wavelength withinwaveguide core 936 according to the following equation:

${{\cos\;\theta} = {\frac{n_{2}}{n_{1}} - {\frac{l}{\Lambda} \cdot \frac{\lambda}{n_{1}}}}},{l = {\pm 1}},{\pm 2},\ldots$where θ is the angle of the incident beam that creates the best overlapintegral with the mode structure of the optical energy propagating inthe waveguide, λ is the wavelength of optical mode propagation down thewaveguide core, n₁ is the refractive index of the waveguide claddingmaterial that includes the gratings, n₂ is the effective refractiveindex of the waveguide core for a propagation mode at wavelength λ, Λ isthe pitch of the gratings in the top cladding layer of the waveguide,and l is a non-zero integer. The period of optical grating 940 can beadjusted during fabrication of the substrate for optimizing the opticalcoupling between the source of optical energy (not shown) and waveguidecore 936.

Such gratings can be made by, e.g., etching periodical features throughthe waveguide cladding along a waveguide core, where the features canbe, e.g., evenly spaced submicrometer sized holes or trenches lined upalong the waveguide. The spacing and size of the features should satisfythe above-described coupling equation. For efficient fabrication of thedevice, the grating can be made on the same mask layer through which theanalyte regions, e.g., nanoholes, are formed. Although describedprimarily in terms of waveguide arrays, such gratings can also be usedto couple optical energy into other types of waveguide substrates, e.g.,those comprising one or more planar waveguides.

Waveguide Arrays for Enhanced Waveguide Illumination Efficiency within aDetection Region of the Array

As opposed to previous illumination strategies where each analyte regionof a substrate requires its own source of optical energy, e.g., a laserbeam, for illumination, waveguide illumination has the advantage ofilluminating a plurality of analyte regions, e.g., hundreds or thousandsof analyte regions, using the power equivalent to a single laser beam.Despite the efficiency advantages of illuminating reaction regions viawaveguides, improvements in waveguide illumination efficiency aredesirable.

The present invention provides waveguide arrays that improve theillumination efficiency of the arrayed waveguides within a detectionregion of the array. The waveguide arrays of the invention include,e.g., optical grating pairs that flank a detection region of the array.Optical grating pairs can be disposed upon an external surface of thearrayed waveguide cores such that optical energy of a desired wavelengthis reinforced within the cores and within a detection region of thearray, e.g., the region of the array in which analyte regions aredisposed proximal to the external surface of the waveguides. Thegrating-mediated reinforcement of optical energy of a desired wavelengthwithin the detection region of the array is advantageous in numerousrespects, e.g., decreasing the power requirements for illuminating thearrayed waveguides while still providing sufficient illumination ofanalyte regions by evanescent fields emanating from the waveguidesduring operation of the device.

FIG. 10 schematically illustrates an example waveguide array thatemploys optical grating pairs for enhanced illumination efficiency,e.g., by enhancing the electric field intensity within the detectionregion without a corresponding increase in the input power level. Asshown, substrate 1000 is provided including a number of branchwaveguides, e.g., surface exposed branch waveguides 1002, 1004, 1006 and1008, that are optically coupled to originating waveguide 1010. Aplurality of reaction regions (not shown) are disposed proximal tobranch waveguides 1002-1008 within detection region 1012. Opticalgratings 1014, 1016, 1018 and 1020, e.g., submicrometer wide holes orslits periodically etched through a waveguide cladding layer (not shown)proximal to each waveguide core, are disposed adjacent to detectionregion 1012 on a first side. Optical gratings 1022, 1024, 1026 and 1028are disposed adjacent to detection region 1012 on a side oppositerelative to the first side. The period of optical gratings 1014-1020 and1022-1028 can be designed (e.g., based on the natural modes ofoscillation of the input light) and fabricated such that optical energyof a desired wavelength is reinforced within detection region 1012 ofthe device, thereby enhancing the illumination efficiency of thewaveguides within detection region 1012 without increasing (andpotentially even decreasing) the power requirements of the device.

The present invention also provides waveguide arrays that improve theillumination efficiency of the arrayed waveguides within a detectionregion of the array by connecting waveguides to recycle the laser power,e.g. at the ends. For example, an end of a first waveguide can beattached to an end of a second waveguide by a bent waveguide, andmultiple waveguides can be so attached in a single waveguide substrate.In some embodiments, an originating waveguide can be split into multiplebranch waveguides, and the distal ends of the branch waveguides can beconnected together. In other embodiments, multiple waveguides can berecombined into a single waveguide, e.g., to recycle laser power. Forexample, as described elsewhere herein, propagation loss causes adecrease in energy density as optical energy is propagated along thelength of the waveguide, e.g., resulting in non-uniform optical energyacross a detection region. The loss in power uniformity can be mitigatedby “recombining” branch waveguides, e.g., within the detection region,to create a merged waveguide having a higher intensity optical energythan either of the branch waveguides, and the capability to propagate adesired energy intensity that is no longer propagated by the branchwaveguides. In yet further embodiments, an originating waveguide is notsplit and is instead arranged in a serpentine manner to cross thedetection region multiple times. In yet further embodiments, acombination of waveguide splitters, recombiners, and/or serpentinearrangements of waveguides is used to provide illumination to thedetection region. The connected waveguides may or may not be adjacent toone another. Further, the waveguides may be connected two-by-two, e.g.using a bent waveguide connector. Alternatively, the waveguides may beconnected with a reverse-splitter-type waveguide, e.g. where a singleconnecting waveguide connects more than two waveguide ends together. Thenumber of relays or the total propagation length is determined by thepropagation loss within each straight section of the branches and theloss in the bending region. The minimum bending radius is determined bythe refractive index contrast of the core and cladding of thewaveguides. The higher the refractive index contrast, the smaller thebending radius. In some embodiments, the bending radius requirement isrelaxed by using three-dimensional connectors to join non-adjacentwaveguides. Further, waveguides in the various configurations describedabove can also be combined with other aspects of the present invention.For example, a branch waveguide and/or a waveguide comprising opticalgrating pairs can be tapered or otherwise modified (e.g., with respectto refractive index, depth, and the like) to promote even illuminationover the detection region, as described elsewhere herein.

FIG. 11 schematically illustrates an example waveguide array in whichthe ends of the arrayed waveguides are connected. As shown, substrate1100 is provided comprising an originating waveguide 1110 that is notsplit, but rather passes repeatedly across a detection region 1112 in aserpentine arrangement. A plurality of reaction regions (not shown) aredisposed proximal to originating waveguides 1110 within detection region1112, all of which are illuminated by a single waveguide core. Althoughthe embodiment in FIG. 11 has a single “serpentine” waveguide core and asingle detection region, a single substrate can comprise one or moreserpentine waveguide cores for illumination of one or more detectionregions. Further, the serpentine waveguide arrangement can also becombined with other aspects of the present invention. For example, aserpentine waveguide can be tapered or otherwise modified (e.g., withrespect to refractive index, depth, and the like) to promote evenillumination over the detection region, as described elsewhere herein.

Waveguide Arrays Comprising Both Optical Splitter and Biosensing Portionon a Single Substrate

The present invention provides waveguide arrays that perform bothoptical splitting and biosensing functions. For example, as describedabove, FIG. 4A provides a top view of one embodiment in which a firstoptical fiber 403 is optically coupled to an originating waveguide 404disposed in substrate 402. Although FIG. 4A illustrates originatingwaveguide 404 being split into all six branch waveguides 406, 408, 410,412, 414, and 416 using a T-splitter conformation, the splitting mayalso occur using a Y-branch splitter conformation, e.g., such that anoriginating waveguide is split into two branch waveguides, which arethen each split to generate a total of four waveguides, two of which arethen split for a total of six waveguides.

Waveguide arrays that perform both optical splitting and biosensingfunctions, while effective, present technical challenges involving,e.g., how optical energy is coupled into the waveguide array. Forexample, in certain preferred embodiments, free space laser light iscoupled into input port(s) of waveguide(s), e.g., from the side of thesubstrate. The small modal profile of the coupled waves, however, cancause a portion of the input optical energy to be coupled into andpropagated through the substrate. Where the optical energy is directedtoward the detection region, this substrate-coupled optical energy cancreate unwanted background noise. Further, optical energy can also belost from the waveguide cores during the splitting process, and such“scattered” optical energy in the substrate can also result in increasedbackground noise, especially when the splitter is located near thedetection region. The present invention provides alternativeconformations or layouts of waveguide arrays comprising both opticalsplitters and biosensing regions that address these potential problemsby mitigating or preventing such substrate-coupled background noise inthe detection region of the substrate.

FIG. 12 illustrates one specific embodiment of a waveguide substrate.Waveguide substrate 1200 comprises Y-branch splitter region 1205 thatsplits three originating waveguides 1210 into a total of 34 branchwaveguides 1215 (2+16+16, although fewer are shown to simplify theillustration.) Optical energy 1220 is coupled into substrate 1200 atinput end surface 1225, both into the cores of originating waveguides1210, as well as into the matrix of waveguide substrate 1200 aroundoriginating waveguides 1210. To prevent increased background noise inbiosensing region 1230 due to optical energy coupling loss at the inputsof originating waveguides 1210 (such as surface scattering, modemismatching, etc.), branch waveguides 1215 are bent at a positionupstream of biosensing region 1230, termed bend region 1235. The bend inthe waveguides ensures that biosensing region 1230 is not in the path ofoptical energy 1220 that is coupled into the matrix of substrate 1200.After passing through biosensing region 1230, optical energy 1220 thatremains in branch waveguides 1215 exits waveguide substrate 1200 atoutput end surface 1240. Although FIG. 12 shows a 90° bend in bendregion 1235, bends of other angles can also be used to remove abiosensing region from the path of optical energy (e.g., excitationradiation) coupled into a waveguide substrate. For example, for an inputcoupling optics with 0.5 N.A. (numerical aperture), an envelope angle ina fused silica substrate is ˜20 degrees. This envelope angle requires atleast a 5.5 mm vertical offset between a biosensing region and awaveguide input ports, if the latter two are 15 mm apart horizontally.To satisfy this condition, the vertical offset can be chosen to be about10 mm, e.g., in the layout in FIG. 12. Further, in certain preferredembodiments, waveguide substrate 1200 further comprises coarse and finealignment marks to align an optical detection system with waveguidesubstrate 1200. For example, the small squares (e.g., 1245) and largesquare 1250 on substrate 1200 can be used as such alignment marks. Incertain preferred embodiments, at least one alignment mark comprisessilicon nitride and is about 2-5 mm².

In certain embodiments, a waveguide substrate such as that illustratedin FIG. 12 has horizontal dimensions on the order of 10-30 mm, and incertain preferred embodiment, such a waveguide substrate isapproximately 20 mm². In certain embodiments, a portion of a waveguidecore that is within a biosensing region is about 1-5 mm long. In certainembodiments, a biosensing region is about 1-10 mm wide. In certainembodiments, there is at least about 1-5 mm clearance between an edge ofa waveguide substrate and the nearest branch waveguide. In certainembodiments, a biosensing region is about 2-10 mm from an output endsurface and about 10-20 mm from an input end surface. In certainembodiments, dimensions are extended to or adjusted based upon a largeror smaller substrate, e.g. a 4, 6, or 8 inch substrate or “wafer” inwhich one or more biosensing regions are placed. Further, although FIG.12 illustrates a configuration with a single bend region and a singlebiosensing region, the invention contemplates substrates with two ormore bend regions and/or biosensing regions. Substrates comprisingmultiple reaction sites are described elsewhere herein, and include,e.g., arrays of nanoholes or zero-mode waveguides, and optionallyintegrated optical detection systems (e.g., lens arrays).

Waveguide Arrays Comprising Separate Optical Splitter and BiosensingSubstrates

Waveguide arrays that perform both optical splitting and biosensingfunctions, while effective, present technical challenges involving,e.g., the fabrication of a single device that performs two disparatefunctions. Splitting optical energy from an originating waveguide into,e.g., 32 or more waveguides, using conventional Y splitters consumesspace on the array, while it is preferable to allocate as much space aspossible to the analytic, e.g., biosensing, portion of the array. Forexample, increasing the multiplex number, e.g., the number of analyteregions, disposed upon a waveguide array is technically challenging whena substantial portion of the array is occupied by features dedicated tosplitting optical energy from an originating waveguide into a pluralityof waveguides. New waveguide arrays that address this issue aretherefore desirable.

The present invention provides devices comprising separate opticalsplitter and biosensing waveguide substrates. Such devices areadvantageous for numerous reasons. For example, the performance of eachwaveguide substrate can be optimized through distinct fabricationprocesses. Further, the device has significant cost benefits as theoptical splitter substrate is reusable, leaving the biosensing substrateas the only consumable waveguide substrate of the device.

FIG. 13 schematically illustrates an example device comprising separateoptical splitter and biosensor waveguide arrays for illuminating aplurality of analytes. As shown, first substrate 1300 is providedincluding a number of branch waveguides, e.g., surface-exposed branchwaveguides 1302-1312. Branch waveguides 1302-1312 are optically coupledto a source of optical energy (not shown) via originating waveguide1314. Second substrate 1316 is provided including a number ofwaveguides, e.g., surface-exposed waveguides 1318-1328. The waveguidesof the second substrate 1316 are optically coupled to the branchwaveguides of the first substrate 1300. As shown, waveguides 1318-1328of the second substrate 1316 are optically coupled to branch waveguides1302-1312 of the first substrate 1300 at coupling regions 1330-1340.Illumination of analytes is accomplished by disposing the analytesproximal to the waveguides of the second substrate 1316 within detectionregion 1342. It will be appreciated that branch waveguides of the firstsubstrate and waveguides of the second substrate can be fewer or greaterin number. For example, 32 or more branch waveguides of the firstsubstrate and 32 or more waveguides of the second substrate arepossible.

As will be appreciated, optical coupling between the branch waveguidesof the first substrate and the waveguides of the second substrate can beaccomplished by a variety of means. For example, optical coupling can beaccomplished by fabricating the waveguides of the second substrate suchthat the cross-sectional area of the waveguides is greater at theoptical coupling location, e.g., near the branch waveguides of the firstsubstrate, than the cross-sectional area of the waveguides at adetection region of the second substrate. The greater cross-sectionalarea at the coupling location facilitates the entry of optical energyinto the waveguides of the second substrate at the coupling location.

Numerous additional coupling mechanisms are available as well, e.g.,disposing optical coupling elements, e.g., a lens or lenses, between thebranch waveguides of the first substrate and the waveguides of thesecond substrate, such that optical energy exiting the branch waveguidesof the first substrate is focused toward a receiving portion of thewaveguides of the second substrate. Optical energy may also be coupledfrom the branch waveguides of the first substrate via an optical gratingdisposed within the waveguides of the second substrate at a positionbetween the branch waveguides of the first substrate and the detectionregion of the second substrate. Coupling may be efficiently achieved bythe use of Holographic Optical Elements (HOE's) which have the advantageof independent tenability for multiple wavelengths. Solid state devicescan bye used for coupling such as programmable phase arrays that canprovide adjustable coupling efficiencies that can be switched on/off andcan also be tuned or adjusted to offset fabrication errors, wavelengthshifts, various beam ualities, etc. Coupling can also be achieved byevanescent field modes, in which the waveguide structure is designed tooverlap the guided modes of adjacent waveguides such that efficientcoupling is achievable in a passive structure with less sensitivity toalignment errors than some other approaches. Other methods of couplingoptical energy between waveguides are known to those of ordinary skillin the art, and includes but is not limited to the use of opticalfibers.

In certain embodiments, a tapered core is used to improve theperformance of a splitter substrate (or splitter portion of a substratecomprising both a splitter and biosensing portion), as well as theefficiency of coupling light from a waveguide with low confinement and alarge modal profile (e.g., in a splitter substrate or portion) to awaveguide with high confinement and a small modal profile (e.g., in abiosensing substrate or portion). As described above, in certainembodiments a biosensing (or detection) region of a substrate preferablyprovides high refractive index contrast between the waveguide core andthe waveguide cladding. Such an embodiment provides a desired level ofillumination confinement in the substrate to provide optical waveshaving a modal diameter in the submicrometer or “few micrometer” range.In general, the thicker the waveguide, the smaller the modal profilesfor optical waves passing through the waveguide. In contrast, a splittersubstrate should preferably provide a low refractive index contrastbetween the waveguide core and waveguide cladding to promote uniformityof light being propagated from an input waveguide into multiple branchwaveguides. As the size of a waveguide decreases, defects from thefabrication process (e.g., roughness of side walls of the waveguide coreat the core-cladding interface) become relatively larger, and theperformance of a splitter or fiber butt coupling is adversely affected,e.g., the splitting uniformity at a 1×2 splitter junction iscompromised. As the size of the waveguide approaches the size of thedefect, the adverse impact becomes greater. As such, a waveguide withhigh confinement has much higher propagation loss than a waveguide withlow confinement, and this propagation loss can be reduced if the guidedoptical wave intensity at the interface can be lowered.

So, in certain preferred embodiments, the biosensing region/substrate ismade with high refractive index contrast waveguides, and the splitterregion/substrate is made with low refractive index contrast waveguides.However, the insertion loss into a waveguide with a small modal profileis relatively high, whether an input waveguide or a branch waveguide.The closer the modal profiles in the waveguide are to those in thefiber, the higher the coupling efficiency and, therefore, the lower thecoupling loss. For example, in the case of input waveguides, the fiberto waveguide butt-to-butt coupling efficiency depends on the modalprofile of the guided waves in the core. With regards to branchwaveguides, a coupling between a low refractive index contrast waveguide(e.g., in a splitter region) and a high refractive index contrastwaveguide (e.g., in a biosensing region) is expected to have a highdegree of insertion loss. To mitigate this effect, waveguide cores canbe tapered so that at the junction of the branch waveguides in thesplitter region/substrate and the branch waveguides in the biosensingregion/substrate the waveguide core dimensions are similar, therebyminimizing any insertion loss at the junction between the splitterregion/substrate and the biosensing region/substrate. For example, thebranch waveguides in a splitter region can be tapered to increase theirdimensions to the dimensions of the core waveguides in the biosensingregion/substrate. Alternatively or in addition, core waveguides in abiosensing substrate (e.g., preferably outside of the detection region)can be tapered to decrease their dimensions to the dimensions of thebranch waveguides in the splitter region/substrate. The tapering can befabricated in either or both the z and/or y direction.

FIG. 14 provides an exemplary schematic cross-sectional view of oneembodiment of a waveguide substrate 1400 comprising an originatingwaveguide core 1402 passing through a splitter region 1404 to createbranch waveguide cores, e.g., branch waveguide core 1406 in a biosensingregion 1408. Tapering of the originating waveguide core 1402 in thesplitter region is shown at 1410. Nanohole 1412 is also shown inbiosensing region 1408, where it is filled with a fluid volume 1414. Thewaveguide cladding 1416 is thickest above the less restrictiveoriginating waveguide core 1402, e.g., in the input and splitter regions1404, and is thinnest above the more restrictive branch waveguide core1406, e.g, in the biosensing region. This difference in claddingthickness mitigates loss of optical energy into the fluid volume 1414,which helps reduce background noise. Various modifications to thisexemplary embodiment are also contemplated, such as combinations withother modifications described herein and known to those of ordinaryskill in the related fields.

Waveguide Arrays with Decreased Bulk Fluorescence and Propagation LossesDue to Back Reflection

When a plurality of nanoholes are disposed through a translucent masklayer and proximal to a surface-exposed waveguide in a substrate, thenanoholes become scattering sources within the waveguide. In certainapplications where fluorescent dye-containing solutions are disposedover the substrate, scattered light can penetrate through thetranslucent mask layer of the substrate and enter the well containingthe fluorescent solution, creating bulk fluorescence. This bulkfluorescence noise can exceed the fluorescent signals of interest withinthe analyte regions, thereby mitigating the effectiveness of thewaveguide substrate. Further, when analyte regions, e.g., in nanoholes,of uniform spacing are disposed proximal to a waveguide core, thenanoholes can create grating effects that result in back reflection inthe waveguide core. This back reflection can result in propagationlosses within the waveguide core.

The present invention provides waveguide substrates that decrease theamount of scattered light that penetrates beyond the mask layer throughwhich nanoholes are formed, and optionally reduce the grating effectsthat result from uniformly disposing nanoholes proximal to thewaveguide. The waveguide substrates provided by the invention caninclude a top mask cladding layer, e.g., a cladding layer disposed upona mask layer that is impenetrable to light such that nanoholes can beformed through both the cladding and mask layers. At locations along thewaveguide where nanoholes are absent, the top mask cladding layerprevents scattered light from penetrating beyond the top surface of thedevice, thereby mitigating bulk fluorescence that results from thescattered light.

Waveguide substrates of the present invention also address issues ofback reflection in the waveguide core. When nanoholes are uniformlyspaced through a mask layer adjacent to a waveguide, the uniformlyspaced nanoholes can create grating effects that cause back reflectionin the waveguide core. This back reflection results in propagationlosses that adversely affect the performance of the device. The presentinvention provides waveguide substrates in which the nanoholes exhibitnon-uniform spacing, e.g., exhibit a random spacing error, tosubstantially eliminate these grating effects.

An example waveguide substrate for reduced bulk fluorescence anddecreased back reflection is schematically illustrated in FIG. 15. Asshown in cross-section, substrate 1500 is provided including a number ofwaveguides, e.g., waveguide 1502. Mask layer 1504 is disposed upon thelayer of the substrate comprising waveguide 1502. Mask cladding layer1506, e.g., a metal layer (e.g., aluminum or chromium), is disposed overmask layer 1504. Apertures, e.g., nanoholes 1508, 1510 and 1512, areformed through mask cladding layer 1506 and mask layer 1504, such that aportion of nanoholes 1508-1512 is at or near the top surface of, and canbe illuminated by an evanescent field emanating from, waveguide 1502.The illumination of analytic processes within the apertures permits theobservation of such processes by detection system 1518.

In the case of top cladding layers made of metal, deposition may beaccomplished through a number of means, including evaporation,sputtering, spin-coating, chemical vapor deposition or the like. Suchprocesses are described in, e.g., U.S. Pat. No. 7,170,050, previouslyincorporated herein by reference in its entirety.

To reduce grating effects that cause back reflection in the waveguides,the spacing between apertures, e.g., distance 1514 between apertures1508 and 1510 and distance 1516 between apertures 1510 and 1512,optionally exhibit a random spacing placement offset, e.g., at leastabout a 1%, 3%, or 5% random spacing placement offset, as compared toapertures with uniform spacing. Essentially, the pitch is modified todecrease the coherent coupling between the apertures that normallyresult in back reflection into the core. Typically, a decrease of atleast about 3-20 dB is sufficient, and it is well within the skill ofthe ordinary practitioner to determine an appropriate waveguide device,e.g., based on the characteristics of the waveguide (e.g., chemicalcomposition, dimensions, etc. of core, cladding, and apertures) and theoptical energy being propagated therein (intensity, wavelength, modestructure, etc.).

Waveguide Arrays with Greater Confinement of Fluorescence Emission Anglefrom Nanoholes

The fluorescent signal emission from a labeled reaction component at thebottom of a nanohole in a waveguide substrate has broad angulardistribution and emits no only toward the bottom of the substrate, butalso toward the top. An objective lens (e.g., of an optical train) canbe positioned to collect signal emissions going in a given direction,e.g., toward the bottom of the substrate, but in a single lens systemthose going in the other direction are not collected. It would bebeneficial to increase the amount of signal collected by the opticalsystem, e.g., by increasing the amount of signal directed toward theoptical train. Further, by confining the angular distribution of thesignal, an objective lens with a smaller numerical aperture (N.A.) canbe used, which can also increase the multiplex capabilities of thesystem. The present invention provides waveguide substrates thatincrease the percent of fluorescent signal emissions that are directedtoward the optical train, thereby increasing the peak intensity of thedetected signal.

In certain preferred embodiment, a metal layer placed on top of thewaveguide cladding serves to reflect upward directed photons back towardthe bottom of the substrate. FIG. 16 illustrates a cross section of sucha waveguide substrate 1600 through a tapered nanohole 1602 and channelwaveguide core 1604. A metal layer 1606 is shown upon the surface of thewaveguide cladding. In this embodiment, the nanohole 1602 penetrates thewaveguide cladding 1608, but does not penetrate the channel waveguidecore 1604. In preferred embodiments, the submicrometer opening at thetop of the nanopore is of a subwavelength diameter to reduce or preventlight passing from the observation volume through this aperture to thearea above the substrate and metal layer. In preferred embodiments, themetal layer need only be thick or opaque enough to reflect light downinto the substrate, e.g., about 40 nm to about 250 nm, or about 100 nmthick. Various types of metal can be used in the layer, including butnot limited to aluminum, gold, platinum, silver, chromium, andcombinations thereof.

Detailed finite-difference time-domain (FDTD) simulations show that theangular emission from a fluorophore at the bottom of the nanohole can beconfined by the presence of the aluminum layer on the top of thesubstrate, thereby increasing the proportion of photons that can becaptured with a single objective positioned below the substrate relativeto an objective with an identical numerical aperture below a substratelacking the aluminum layer. The simulations show that the emissionangles are more confined within smaller cones for dipoles polarizedalong either the x or z direction (directions parallel to the top of thesubstrate). For dipole emitters lined up along the y direction, lessangular confinement is provided by the addition of an aluminum layer.Thus, deposition of a reflective layer (such as aluminum) above the topcladding layer of a waveguide chip will enhance the fluorescenceemission intensity and confine the angular distribution. As such,although autofluorescence noise from the waveguide core may be somewhathigher, the benefits in increased emission intensity and confinement ofthe angular distribution are expected to more than compensate for anyincreased background noise.

Waveguide Arrays with Improved Illumination Efficiency

When a plurality of nanoholes are disposed on a substrate and filledwith a fluid (e.g., reaction mixture), the change in refractive index atthe nanoholes can perturb the propagation of the optical waves in thewaveguide, and a large portion of the light can be scattered inundesirable directions. The present invention provides waveguide arraysthat reduce such scattering of propagating light by including “dummynanoholes” spaced closer than the wavelengths of optical wavespropagating within the waveguide core.

The scattering properties of a waveguide optically coupled to an arrayof nanoholes can be described as an output coupler that couples a guidedconfined mode with a propagation constant β to a radiation mode. FIG.17, a longitudinal phase matching diagram illustrating corrugatedwaveguide output coupler, shows the radiation mode escapes from awaveguide core 1702 having a refractive index n₂ at an angle θ into asemi-infinite upper layer 1704 having a refractive index n₁. Forradiation mode into the upper layer 1704, the grading period Λ mustsatisfy Equation 1:

${{\beta - {\frac{\omega\; n_{1}}{c}\cos\;\theta}} = {l\frac{2\pi}{\Lambda}}},{l = {\pm 1}},{\pm 2},\ldots$Using the relationship β=2π/λg and λ=c/f, the above equation can berewritten as Equation 2:

${{\cos\;\theta} = {\frac{n_{2}}{n_{1}} - {\frac{l}{\Lambda} \cdot \frac{\lambda}{n_{1}}}}},{l = {\pm 1}},{\pm 2},\ldots$To have a valid solution for Equation 2, the pitch of the grating or thepitch of the nanoholes (e.g., 1706) must satisfy Equation 3:

$\Lambda \geq \frac{\lambda}{n_{2} - n_{1}}$where the equal sign corresponds to the case θ=0, or the free spaceradiation mode propagates along the direction of the guided mode.

For radiation mode into the semi-infinite lower layer 1708 having arefractive index n₃, using the same derivation as above, the pitch ofgrating must satisfy the following condition to have a radiation mode(Equation 4):

$\Lambda \geq \frac{\lambda}{n_{2} - n_{3}}$For example, the effective refractive indices of a waveguide withnanoholes are n₁=1.33, n₂=1.53, n₃=1.46. For guided optical waves at 532nm, the minimum pitches that can generate free space radiations intoeither the upper or the lower layers are 2.66 μm and 7.6 μm, based onEquation 2 and Equation 4, respectively.

The nanoholes extending into a waveguide substrate act as a periodicalstructure that couples light out into the upper or lower layers. Tosuppress the free space radiation modes from nanoholes, the pitch of thenanoholes is made smaller than the numbers calculated based on Equation3 and Equation 4. If the pitch of the nanoholes is smaller than theresolving power of the imaging optics, dummy nanoholes with identicalrefractive index can be made to suppress the scattering effects. Usingthe 532 nm mode example calculation above, if the optical resolution ofthe imaging optice is 4 μm, nanoholes can be constructed with 2 μmspacing and every other nanoholes can be rendered inaccessible toanalytes by filling them with material having a refractive indexidentical to that of the fluid that will be introduced to the other halfof the nanoholes.

It will be readily understood by one of ordinary skill in the art thatthe examples provided above are for small refractive contrast waveguidestructures. The minimum pitches are much smaller (subwavelength) forwaveguide structures with much higher contrasts. For further informationon periodic structures in integrated optics, see Yariv, et al. (1977)“Periodic Structures for Integrated Optics,” IEEE Journal of QuantumElectronics, Vol. QE-13, No. 4, the disclosure of which is incorporatedby reference herein in its entirety for all purposes.

Waveguide Arrays with Improved Analyte Immobilization Properties

The waveguide arrays of the invention include analyte regions thatoptionally include one or more analytes disposed within the analyteregions. For reliable observation of the analyte by a detection system,it is preferable to immobilize the analyte to a surface of the substratethat is in sufficient proximity to a waveguide core such that theanalyte is illuminated by an evanescent field emanating from thewaveguide core. Targeted immobilization to a surface of a waveguidearray proximal to a waveguide, e.g., proximal to an exposed surface of awaveguide, such that self-alignment of the analytes with the waveguidepattern is achieved, e.g., a near-perfect array of detection spots, isparticularly desirable.

A device of the present invention is schematically illustrated in FIG.18. Substrate 1800 is provided, including one or more waveguides, e.g.,surface-exposed waveguides 1802, 1804, 1806, 1808, 1810 and 1812, whichare optically coupled to originating waveguide 1814. An array ofsubstantially parallel lines of a surface immobilization compound, e.g.,lines 1816, 1818, 1820, 1822 and 1824 are deposited upon substrate 1800such that lines 1816-1824 are substantially perpendicular to waveguides1802-1812. In preferred aspects, lines 1816-1824 are deposited uponsubstrate 1800 such that the lines are deposited upon a top surface ofwaveguides 1802-1812. A mask layer (not shown) can be provided, suchthat only the intersections between lines 1816-1824 and top surface ofwaveguides 1802-1812 are exposed. Analytes (not shown) with an affinityfor the particular material from which the lines are made, e.g., a metal(e.g., gold), is then provided and immobilized at the intersection oflines 1816-1824 and top surface of waveguides 1802-1812.

Deposition can be accomplished by a variety of methods, e.g.,microcontact printing. Alternatively, the metal lines can be deposited,and biased chemistry can be used to situate the analytes on the linesand not in the spaces in between.

Optical Trains and Detection Systems

Optical trains and detection systems for use in carrying optical energy(e.g., illumination) to and/or collecting emitted optical energy from ananalyte region disposed on a waveguide substrate of the inventiongenerally include an optical energy source, e.g., one or more lasers, awaveguide to provide optical energy to one or more analyte regions, anoptical train that transmits emissions so that they can be detected andanalyzed, and detection and data processing components for detecting,storing and presenting signal information. For example, certainembodiments of optical systems useful with the waveguide substratesprovided herein include those described in U.S. Patent Publication No.2008/0128627, which is incorporated herein by reference in its entiretyfor all purposes. Other optical trains and detection systems for usewith waveguide substrates are known to those of ordinary skill in theart, and are provided, e.g., in U.S. Provisional Patent Application No.61/223,628, filed Jul. 7, 2009; U.S. Pat. Nos. 6,437,345, 5,677,196, and6,192,168; U.S. Patent Publication Nos. 2002/0146047, 2007/0188746,2007/0036511, 2005/0175273, and 2008/0030628; and in variouspublications, including Bernini, et al. (2005) Proceedings of SPIE, Vol.5728: 101-111; Boriarski, et al. (1992) Proceedings of SPIE, Vol.1793:199-211; Feldstein, et al. (1999) Journal of BiomedicalMicrodevices, Vol. 1:139-153; Herron, et al. (2003) In: Biopolymers atInterfaces, 2nd Edition (M. Malmsten, Ed.), Surfectant Science Series,Vol. 110, Marcel Dekker, New York, pp. 115-163; and Weissman, et al.(1999) Proceedings of SPIE, Vol. 3596: 210-216, the disclosures of whichare incorporated herein by reference in their entireties for allpurposes. In particular, in certain aspects, the imaged signal will be aseries of discrete signal sources or points of signal origin on theoverall surface of the waveguide substrate. As such, in certain aspectsthe detection systems described in the aforementioned applications aredirectly applicable to the present invention.

One example of a system for use in the present invention is illustratedin FIG. 19. As shown, the system 1900 includes a waveguide substrate ofthe invention 1902. Laser 1904 and optional additional laser 1906 areoptically coupled to one or more waveguides within the substrate, e.g.,via optical fibers 1908. An optical train is positioned to receiveoptical signals from the substrate and typically includes an objective1910, and a number of additional optical components used in thedirection, filtering, focusing and separation of optical signals. Asshown, the optical train includes a wedge prism 1912 for separatingspectrally different signal components, and a focusing lens 1914 thatimages the signal components upon an array detector, e.g., EMCCD 1916.The detector is then operatively coupled to a data storage andprocessing system, such as computer 1918 for processing and storage ofthe signal data and presentation of the data in a user desired format,e.g., on printer 1920. As will be appreciated, a number of othercomponents may be included in the systems described herein, includingbut not limited to mirrors, gratings, switches, and optical filters forfiltering background illumination or bleed-through illumination from theoptical energy sources, from the actual optical signals. Additionally,alternate optical trains may employ cascaded spectral filters inseparating different spectral signal components.

While illustrated with a first optical energy source, e.g., laser 1904,and an optional second optical energy source, e.g., optional laser 1906,it will be appreciated that additional optical energy sources may beprovided optically coupled to the waveguide substrates, e.g., usingadditional originating waveguides to direct light from each the varioussources to all or a subset of the waveguides in a given waveguide array.For example, in some cases, 3 light sources, 4 light sources or more maybe used. Additional light sources will preferably provide light havingdifferent spectral characteristics, e.g., peak wavelengths, to thewaveguides, although they may also be employed to provide additionalintensity or variations in other light characteristics, such asfrequency.

While illustrated with an optical fiber coupling, optical energy canalso be coupled into the waveguide by other means, e.g., using afree-space methodology. For example, optical energy can be coupled intoa waveguide substrate at an edge of the substrate. In certain preferredembodiments, such optical energy is directed at the end of one or morewaveguides and coupled therein. In other embodiments, the optical energyis instead coupled into the waveguide substrate from the side of thesubstrate rather than being directed at the ends of the waveguides. Ineither case, coupling can be achieved through the use of a grating orbutt coupling, both of which are routine in the art.

The detection system is typically configured to detect signals fromlarge areas of the waveguide substrate, e.g., multiple signals emanatingfrom a plurality of different analyte regions on the substrate, andpreferably, do so simultaneously. Thus while scanning detection opticsmay be employed for certain applications of the invention, in general,larger area imaging detection systems are preferred.

In certain embodiments, there are a plurality objective lenses in anoptical system of the invention. For example, one or more objectivelenses may be positioned below the waveguide array and/or one or moreobjective lenses may be positioned above the waveguide array. Suchmultiple objective configurations are useful for both increasing thecollection efficiency and multiplex capabilities of the system.

As described above in the section entitled “Waveguide Arrays withGreater Confinement of Fluorescence Emission Angle from Nanoholes,” if asingle objective lens is positioned under the waveguide substrate,signal emissions that emerge from the top of the substrate are notcollected. In certain embodiments, a plurality of objective lenses areused to mitigate the resulting loss of signal and thereby increasecollection efficiency. An illustrative example of such an embodiment isprovided in FIG. 20, which depicts a cross-section of a waveguidesubstrate 2000 having nanoholes 2002 and channel waveguides 2004. Twoobjective lenses are used: a first objective lens 2006 positioned underthe waveguide array to collect signal emissions that are directed towardthe bottom of the substrate 2000, and a second 2008 positioned above thewaveguide array to collect signal emissions that are directed toward thetop of the substrate 2000. The two objective lenses (2006 and 2008) havethe same field-of-view (FOV) delineated by the double-arrow 2010, andthis FOV defines the detection region 2014. The emission signalscollected by these two objective lenses can be combined with standardoptical train components to be directed to a single detector (e.g.,camera), or may be detected separately, e.g., using two detectors. A twocamera system allows cross-correlation between signal detected at thetop and bottom of a given nanohole, and the additional data so generatedcan increase the accuracy of the system. Statistical analyses forprocessing signal data including cross-correlations are well known tothe ordinary practitioner and routinely practiced in the art.

Additional benefits can also be realized by a multi-objectiveconfiguration. One advantage to waveguide illumination is that theillumination light is confined near the vicinity of the focal plane ofthe imaging optics, so autofluorescence does not tend to scale with themultiplex number, which facilitates observation of higher numbers ofreactions on a single substrate (“higher multiplex”) as compared toother types of illumination that are more prone to autofluorescentbackground signals. However, the collection optics can limit the extentof multiplex detection on a waveguide substrate. For example, in asingle objective lens system a detection region on a substrate can belimited to the size of the FOV of the objective lens. To increase themultiplex, a custom objective lens may be designed and constructed toimage a larger field-of-view, but such design and construction are bothtime-consuming and expensive. A more economical solution provided by theinstant invention is to double the multiplex by using two separateoff-the-shelf objective lenses, which are less expensive and morereadily available than a custom objective lens would be. An illustrativeexample of such an embodiment is provided in FIG. 21, which depicts across-section of a waveguide substrate 2100 having nanoholes 2102 andchannel waveguides 2104. As for the double lens system described above,there is one objective 2106 positioned under the waveguide substrate anda second objective 2108 positioned over the waveguide substrate.However, in contrast with the two objective lens system described above,the objective lenses 2106 and 2108 are not aligned on top of each other,but rather offset from one another so that each detects emissions from adifferent portion of the detection region 2114. For example, the firstobjective 2106 has an FOV delineated by the double arrow 2112, and socollects signal from a first half of the detection region 2118, and thesecond objective 2108 has an FOV delineated by the double arrow 2110,and so collects signal from a second half of the detection region 2116.As such, an area with twice the FOV of each individual objective lenscan be monitored and imaged, one FOV by the objective positioned at thebottom of the substrate, and one FOV by the objective positioned at thetop of the substrate. Alternatively or in addition, more than oneobjective lens could be positioned on the same side of the substrate,e.g., if the substrate comprised more than one discrete and separatedetection region and they were positioned within the FOVs of theadjacent objective lenses, e.g., given the size of the lens housings andany other structures required to position the lenses. Further, an arrayof objective lenses could be used to detect signal from a large singledetection region, with some objective lenses above the substrate andsome below, so long as the FOVs of the arrayed lenses covered the entiredetection region. As such, using two objectives can thereby increase themultiplex number by two-fold as compared to only one objective, and eachadditional objective on the top or bottom of the substrate can provideanother fold-increase in the multiplex capabilities of the system.Further, by using off-the-shelf objective lenses rather than large,expensive custom lenses, a cost savings is realized, as well.

In certain preferred embodiments, one or more microlens arrays arecomponents of an optical system that is integrated into a device forsingle-molecule (e.g., single-reaction-site) detection. In certainaspects, such a device is a single unit that contains multiple layers: asubstrate comprising one or more nanoholes or ZMWs, lens arrays,gratings (e.g., Fresnel wedge gratings), and sensors. One or morenanoholes or ZMWs may optionally be disposed within a confinement on thesurface of the substrate, e.g., a well or channel. In typicalimplementation, the layers of the device are fabricated separately bydifferent process that achieve the specific specification requirementsfor each layer. After fabrication, the layers are aligned with oneanother during assembly of the device. Manufacture and precise alignmentof the layers can be achieved by known methods, e.g., based onconventional semiconductor or microarray fabrication processes, and soare within the level of one of ordinary skill in the art.

FIG. 22 provides a cross-sectional view of an illustrative embodiment ofsuch an integrated device 2200 in which nanoholes 2202 (or ZMWs) arefabricated on top of a planar or channel waveguide 2204 in which opticalenergy 2206, e.g., illumination light, is propagated. A first microlensarray 2208 is positioned beneath the waveguide layer near the nanoholes(or ZMWs), and the microlenses therein can be fabricated at a micrometerpitch with a larger numerical aperture than, e.g., a single objectivelens positioned under the waveguide intended to capture light from allthe arrayed nanoholes (or ZMWs). In certain embodiments, each of themicrolenses collects the photons (depicted as diverging arrows, e.g.,2210) emitted from a single nanohole (or ZMW) and sends a collimatedbeam (e.g., 2212) downward. The collimated beam, e.g., of fluorescentlight, passes through a notch filter layer 2214 that rejects unwantedscattering light and/or autofluorescence noise. A second microlens arraylayer 2216 is positioned in front of a detector to focus the collimatedlight onto each pixel 2218 of the detector. To reduce the need forcomplex, spectral-splitting, free-space optical components, such as awedge or multichannel dichroic filter, the system can also use a singlecolor mode in which the excitation radiation is gate, with theexcitation lasers working at a pulse mode that matches the detectiongating. Therefore, a spectral splitter would not be needed because eachdifferent wavelength of optical energy would be propagated though thewaveguide in a temporally separate manner, e.g., one at a time. (Methodsfor pulse mode excitation are provided, e.g., in U.S. Patent PublicationNo. 20090181396, incorporated herein by reference in its entirety forall purposes.) As such, this optical system provides a set ofmicrometer-scale imaging optics for each nanohole (or ZMW). The pitch ofthe microlens arrays can be chosen to match the pitch of the pixels on adetector. With essentially no limitation on the FOV, the multiplexcapabilities of the optical system are vastly increased over moretraditional free-space optical systems previously used with ZMW arrays.

FIG. 23 provides a further embodiment of a device comprising anintegrated optical system with microlens arrays. Three major componentsof device 2300 are shown in the cross-sectional representation: (1) awaveguide substrate layer 2302, (2) a wafer-level lens array layer 2304,and (3) a sensor array layer 2306. The waveguide substrate layer 2302comprises a plurality of reagent wells 2308, each of which comprises abiosensing region (e.g., 2310) with an array of nanoholes (or ZMWs). Thewaveguide 2312 (e.g., core and cladding) is located in the waveguidesubstrate layer 2302, and may be a planar waveguide, or a series ofchannel waveguides, as described elsewhere herein. The waveguide 2312delivers optical energy to reaction sites in the biosensing regions(e.g., 2310), e.g., at the bottom of the nanoholes (or ZMWs). Thewafer-level lens array layer 2304 comprises multiple “mini objectivelenses” formed from several layers of microlenses, and is positionedunder the waveguide substrate layer 2302. To reach high numericalaperture for increased collection efficiency, an immersion fluid layer2314 can optionally be implemented to optically connect the waveguidesubstrate layer 2302 and the wafer-level lens array layer 2304.Dielectric coatings (e.g., dielectric notch filters 2316) to block laserlight can be integrated into the wafer-level lens array, and dispersivegratings 2318 (or Fresnel wedge(s)) can also be integrated at the waferlevel to spread light and facilitate detection. For example, fluorescentlight of differing wavelengths can be spread to facilitate detection ofthe individual wavelengths. The sensor array layer 2306 (e.g.,comprising CMOS, CCD, etc.) is positioned under the wafer-level lensarray layer 2304, and comprises multiple discrete sensor arrays 2320,each of which is aligned with one of the mini objective lenses in thewafer-level lens array layer 2304. Each of the sensor arrays 2320 imagesemission signals from nanoholes (or ZMWs) within a given reagent well.As will be clear, the device depicted in FIG. 23 is merely one exampleof an integrated optical system device of the invention, and othervariations and substitutions on this illustrative example arecontemplated. For example, such devices may comprise additionalmicrolens layers, different layouts, different types of opticalcomponents (e.g., gratings, mirrors, lenses, couplings, filters, etc.),and the like.

Since all the layers a device comprising integrated optical components(e.g., as described above) can be made at the waveguide substrate level,the device can be to be scaled to extremely high multiplex. For example,a waveguide substrate could comprise a 10×10 array of parallel reagentwells, with each well having dimensions of about 1×1 mm and containingapproximately 32,000 nanoholes (or ZMWs). The total multiplex of thisexemplary device is 3.2 million; i.e., 3.2 million separate analytes oranalytical reactions can be individually and simultaneously monitored inreal time with such a device. This level of multiplex far exceeds whatconventional free space optics typically achieves. A further advantageover systems utilizing free space optics is that alignment of themultiple lens arrays in the integrated optics devices is readilyachieved using standard microlithography techniques, and once assembledthe device is far less sensitive to vibration or thermal drift. Inaddition, the advantages of waveguide illumination over free spaceillumination also apply and include, e.g., spatially confinedautofluorescence, lower input power requirements, smaller size andweight, and lower costs for manufacturing, packaging, and the like.

In certain preferred embodiments, one or more multilayer dielectricstacks that have been tuned to have particular reflectance propertiesare components of an optical system that is integrated into a device forsingle-molecule (e.g., single-reaction-site) detection. One example ofsuch a tuned dielectric stack is a dielectric omnidirectional reflector(or “mirror”). In preferred embodiments, the reflectance propertiesinclude reflection over a wide range of angles and polarizations forparticular wavelengths (e.g., excitation illumination wavelengths)combined with permission of other wavelengths (e.g., emissionwavelengths). Although not technically a “waveguide” as describedelsewhere herein, a dielectric reflector serves a function similar tothat of a waveguide in a waveguide substrate. However, while waveguidesubstrates are typically angle selective with regards to containment ortransmission of optical energy, dielectric reflectors are typicallywavelength selective and can be fabricated to reflect a first set ofwavelengths (e.g., excitation illumination wavelengths) while allowingpassage of a second set of wavelengths (e.g., emission illuminationwavelengths). In certain aspects, such a device comprises multiplelayers: a substrate comprising one or more nanoholes or ZMWs, a masklayer over the surface of the substrate, and a dielectricomnidirectional reflector under the substrate. Dielectricomnidirectional reflectors are known in the art, e.g., in Deopura, etal. (2001) Optics Letters 26(15):1197-1199; and Fink, et al. (1998)Science 282:1679-1682, both of which are incorporated herein byreference in their entireties for all purposes. In certain preferredembodiments, a dielectric omnidirectional reflector comprises a stack ofdielectric layers that are configured to reflect optical energy from anenergy source at one or more excitation wavelengths, while permittingtransmission of optical energy emitted from nanoholes or ZMWs (emissionradiation) to an optical detection system. The mask layer comprisesmaterial that reflects optical energy, and in particular excitationradiation from the optical energy source; in preferred embodiments, atleast the portion of the mask layer in contact with the substrate is ametal (e.g., aluminum, gold, silver, platinum, and the like) thatreflects essentially all the excitation radiation back down into thesubstrate layer. The trapping of the optical energy within the substratelayer can be adjusted or “tuned” by methods known to the skilledpractitioner to achieve a desired level and/or wavelength(s) ofreflection of the optical energy, and the desired reflection is chosenbased on various factors including, but not limited to, a tolerance ofthe system to autofluorescence generated by such reflection, and thequantity of heat that can be dissipated from the device. One or morenanoholes or ZMWs may optionally be disposed within a confinement on thesurface of the substrate, e.g., a well or channel. In typicalimplementation, the layers of the device are fabricated separately bydifferent processes that achieve the specific specification requirementsfor each layer. After fabrication, the layers are aligned with oneanother during assembly of the device. Manufacture and precise alignmentof the layers can be achieved by known methods, e.g., based onconventional semiconductor or microarray fabrication processes, and soare within the level of one of ordinary skill in the art.

FIG. 24 provides an illustrative example of a preferred embodiment ofthe invention. Device 2400 comprises an optical energy source 2410(e.g., a laser, light emitting diode, or other narrow emission source)delivers optical energy 2420 (e.g., of one or more excitationwavelengths) to the edge of substrate 2430 where it passes into thesubstrate to be propagated between mask layer 2440 and dielectricomnidirectional reflector 2450. Dielectric omnidirectional reflector2450 reflects optical energy 2420 into substrate 2430, but permitspassage of optical energy 2460 emitted from nanoholes or ZMWs 2470(emission radiation) through the reflector 2450 to an optical detectionsystem 2480.

The use of a dielectric omnidirectional reflector to propagate opticalenergy in a single-molecule detection device provides many of the samebenefits as use of waveguide illumination, including mitigation ofmisalignment of an optical energy source and a biosensing region(s) on asubstrate. In addition, an omnidirectional dielectric reflector may beintegrated within a substrate, placed in direct contact with asubstrate, or may be positioned such that a layer of air (or other gas,fluid, etc.) separates the reflector from the substrate. Further, suchdevices can be used to illuminate various types of reaction sites, e.g.reaction sites located within nanoholes or zero-mode waveguides, orillumination of other types of analytical reaction systems known in theart.

Those of ordinary skill in the art will understand that various changesin form and detail can be made to the substrates, waveguides, dielectricreflectors, and nanoholes provided herein. For example, variation of thenanohole geometry can vary the optical field produced within thewaveguide core, as well as the observation volume being illuminated bythe field. Further, different waveguide geometries can be used todeliver excitation radiation to the nanoholes, including variousarrangements of channel waveguides and/or planar waveguides, some ofwhich are described elsewhere herein. In particular, single analytes,molecules, molecular complexes can be detected, monitored, and analyzedin real time, e.g., during the course of an analytical reaction.

III. Methods and Applications

As noted previously, the substrates, systems and methods of theinvention are broadly applicable to a wide variety of analyticalmethods. In particular, the waveguide substrates of the invention may beemployed in the illumination-mediated analysis of a range of materialsthat are disposed upon or proximal to the substrate's surface. Suchanalyses include, inter alia, a number of highly valued chemical,biochemical and biological analyses, including nucleic acid analysis,protein interaction analysis, cellular biology analysis, and the like.

Exemplary Applications

1. Sequencing by Synthesis

One example of an analytical operation in which the present invention isparticularly applicable is in the determination of nucleic acid sequenceinformation using sequence-by-synthesis processes. Briefly,sequencing-by-synthesis exploits the template-directed synthesis ofnascent DNA strands, e.g., using polymerase-mediated strand extension,and monitors the addition of individual bases to that nascent strand. Byidentifying each added base, one can deduce the complementary sequencethat is the sequence of the template nucleic acid strand. A number of“sequence-by-synthesis” strategies have been described, includingpyrosequencing methods that detect the production of pyrophosphate uponthe incorporation of a given base into the nascent strand using aluminescent luciferase enzyme system as the indicating event. Becausethe indicator system is generic for all four bases, the process requiresthat the polymerase/template/primer complex be interrogated with onlyone base at a time.

Other reported sequence-by-synthesis methods employ uniquely labelednucleotides or nucleotide analogs such that the labels provide both anindication of incorporation of a base, as well as indicate the identityof the base (See, e.g., U.S. Pat. No. 6,787,308, incorporated herein byreference in its entirety for all purposes). Briefly, these methodsemploy a similar template/primer/polymerase complex, typicallyimmobilized upon a solid support, such as a planar or other substrate,and interrogate it with nucleotides or nucleotide analogs that mayinclude all four bases, but where each type of base bears an opticallydetectable label that is distinguishable from the other bases. Thesesystems employ terminator bases, e.g., bases that, upon incorporation,prevent further strand extension by the polymerase. Once the complex isinterrogated with a base or mixture of bases, the complex is washed toremove any non-incorporated bases. The washed extended complex is thenanalyzed using, e.g., four color fluorescent detection systems, toidentify which base was incorporated in the process. Followingadditional processing to remove the terminating group, e.g., usingphotochemistry, and in many cases, the detectable label, the process isrepeated to identify the next base in the sequence. In some cases, theimmobilized complex is provided upon the surface as a group ofsubstantially identical complexes, e.g., having the same primer andtemplate sequence, such that the template mediated extension results inextension of a large number of identical molecules in a substantiallyidentical fashion, on a step wise basis. In other strategies, complexesare immobilized in a way that allows observation of individual complexesresulting in a monitoring of the activity of individual polymerasesagainst individual templates.

As will be appreciated, immobilization or deposition of thepolymerase/template/primer complex upon or proximal to the surface ofthe waveguide core in the waveguide arrays of the invention will allowillumination, and more notably in the case of fluorescence-based assays,excitation, at or near selected regions of the surface without excessiveactivation and fluorescence interference from the surroundingenvironment, which can be a source of significant noise in fluorescencebased systems.

In another sequencing-by-synthesis process, one monitors the stepwiseaddition of differently labeled nucleotides as they are added to thenascent strand and without the use of terminator chemistries. Further,rather than through a one-base-at-a-time addition strategy, monitoringof the incorporation of bases is done in real time, e.g., without theneed for any intervening wash steps, deprotection steps or separatede-labeling steps. Such processes typically rely upon optical strategiesthat illuminate and detect fluorescence from confined reaction volumes,such that individual complexes are observed without excessiveinterference from labeled bases in solution that are not beingincorporated (See U.S. Pat. Nos. 6,991,726 and 7,013,054, previouslyincorporated herein, and U.S. Pat. Nos. 7,052,847, 7,033,764, 7,056,661,and 7,056,676, the full disclosures of which are incorporated herein byreference in its entirety for all purposes), or upon labeling strategiesthat provide fluorescent signals that are indicative of the actualincorporation event, using, e.g., FRET dye pair members on a base and ona polymerase or template/primer (See U.S. Pat. Nos. 7,052,847,7,033,764, 7,056,661, and 7,056,676, supra).

In accordance with the foregoing sequence-by-synthesis methods, one mayoptionally provide the complexes over an entire surface of a substrate,or one may selectively pattern the immobilized complexes upon orproximal to the waveguide cores. Patterning of complexes may beaccomplished in a number of ways using selectively patternable chemicallinking groups, and/or selective removal or ablation of complexes not inthe desired regions. In some cases, one can employ the waveguides inselectively patterning such complexes using photoactivatable chemistrieswithin the illumination region. Such strategies are described in detailin U.S. patent application Ser. No. 11/394,352 filed Mar. 30, 2006, thedisclosure of which is incorporated herein by reference in its entiretyfor all purposes.

In addition to selective immobilization, and as noted above, in somecases it is desirable to immobilize the complexes such that individualcomplexes may be optically resolvable, e.g., distinguished from othercomplexes. In such cases, the complexes may be immobilized using highlydilute solutions, e.g., having low concentrations of the portion of thecomplex that is to be immobilized, e.g., the template sequence, thepolymerase or the primer. Alternatively, the surface activation forcoupling of the complex component(s) may be carried out to provide a lowdensity active surface to which the complex will be bound. Such surfaceshave been described in U.S. patent application Ser. No. 11/240,662,filed Sep. 30, 2005, which is incorporated herein by reference in itsentirety for all purposes. Again, such low density complexes may bepatterned just upon or proximal to the waveguides or they may beprovided across the surface of the substrate, as only those reactioncomplexes that are proximal to the waveguides will yield fluorescentsignals.

While described in terms of real-time nucleic acidsequencing-by-synthesis, it will be appreciated that a wide variety ofreal-time, fluorescence based assays may be enhanced using the waveguidearrays and methods of the invention. In particular, the waveguide arraysystems provided herein facilitate simultaneous illumination anddetection of multiple fluorophores of multiple different wavelengths isreal time for a variety of experimental systems.

2. Molecular Arrays and Other Surface Associated Assays

Another exemplary application of the waveguide arrays of the inventionis in molecular array systems. Such array systems typically employ anumber of immobilized binding agents that are each specific for adifferent binding partner. The different binding agents are immobilizedin different known or readily determinable locations on a substrate.When a fluorescently labeled material is challenged against the array,the location to which the fluorescently labeled material binds isindicative of its identity. This may be used in protein-proteininteractions, e.g., antibody/antigen, receptor-ligand interactions,chemical interactions, or more commonly in nucleic acid hybridizationinteractions. See, U.S. Pat. Nos. 5,143,854, 5,405,783 and relatedpatents, and GeneChip® systems from Affymetrix, Inc.

In accordance with the application of the invention to arrays, a numberof binding regions, e.g., populated by known groups of nucleic acidprobes, are provided upon a substrate surface upon or proximal to thewaveguides such that a hybridized fluorescently labeled probe will fallwithin the illumination region of the waveguide. By providing forselective illumination at or near the surface, one can analyzehybridized probes without excessive interference from unboundfluorescent materials. Further details regarding this aspect of theinvention can be found in Lundquist et al. U.S. Patent Publication No.2008/0128627, the full disclosure of which is incorporated herein byreference in its entirety for all purposes.

3. Cellular Observation and Analysis

In still another exemplary application, cell-based assays or analysesmay be carried out by providing cells adhered to the substrate surfaceover the waveguides. As a result, one could directly monitorfluorescently labeled biological functions, e.g., the uptake offluorescent components, the generation of fluorescent products fromfluorogenic substrates, the binding of fluorescent materials to cellcomponents, e.g., surface or other membrane coupled receptors, or thelike.

4. Other Applications

It will be appreciated by those of ordinary skill that the substrates ofthe invention may be broadly applicable in a wider variety ofapplications that monitor analytical processes, including but notlimited to those provided in U.S. Patent Application Nos. 61/186,645 and61/186,661, both of which were filed Jun. 12, 2009 and are incorporatedherein by reference in their entireties for all purposes. In addition,such substrates and methods may be employed in the identification oflocation of materials on surfaces, the interrogation of quality of agiven process provided upon the surface, the photo-manipulation ofsurface bound materials, e.g., photo-activation, photo-conversion and/orphoto-ablation. As such, while some of the most preferred applicationsof the present invention relate to analytical operations andparticularly in the fields of chemistry, biochemistry, molecular biologyand biology, the discussion of such applications in no way limits thebroad applicability of the invention.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it is to be understood that theabove description is intended to be illustrative and not restrictive. Itwill be clear to one skilled in the art from a reading of thisdisclosure that various changes in form and detail can be made to theinventions disclosed herein without departing from the true scope andspirit of the invention. For example, all the techniques and apparatusdescribed above can be used in various combinations. The scope of theinvention should, therefore, be determined not with reference to theabove description, but should instead be determined with reference tothe appended claims, along with the full scope of equivalents to whichsuch claims are entitled. All publications, patents, patentapplications, and/or other documents cited in this application are citedfor the purpose of describing and disclosing reagents, methodologies andconcepts that may be used in connection with the present invention.Nothing herein is to be construed as an admission that these referencesare prior art in relation to the inventions described herein. Throughoutthe disclosure various patents, patent applications and publications arereferenced. Unless otherwise indicated, each is incorporated herein byreference in its entirety for all purposes to the same extent as if eachindividual publication, patent, patent application, and/or otherdocument were individually indicated to be incorporated by reference forall purposes.

What is claimed is:
 1. An integrated optical system comprising: awaveguide comprising a cladding layer and an optical waveguide layer; aplurality of nanoholes in the cladding layer; a microlens array; and adetector comprising a plurality of sensing regions; wherein theplurality of nanoholes and the microlens array are positioned onopposite sides of the optical waveguide, wherein the plurality ofnanoholes are positioned to be illuminated by an evanescent fieldemanating from the optical waveguide when optical energy is propagatedtherethrough, and wherein the microlens array is configured to collectoptical energy signals emitted from each of the plurality of nanoholesand send the collected optical energy signals to the plurality sensingregions of the detector.
 2. The integrated optical system of claim 1,wherein the plurality of nanoholes penetrate a core of the opticalwaveguide.
 3. The integrated optical system of claim 1, wherein thenanoholes are zero-mode waveguides.
 4. The integrated optical system ofclaim 1, wherein the optical waveguide is a planar waveguide.
 5. Theintegrated optical system of claim 1, wherein the optical waveguidecomprises at least one channel waveguide.
 6. The integrated opticalsystem of claim 1, wherein the microlens array comprises a firstmicrolens array and a second microlens array, wherein the firstmicrolens array is configured to collect emitted optical energy fromeach of the plurality of nanoholes and send a collimated beam of theemitted optical energy to the second mircolens array, wherein the secondmicrolens array is configured to send the collimated beam of emittedoptical energy from each of the plurality of nanoholes to the pluralityof sensing regions of the detector.
 7. The integrated optical system ofclaim 6, further comprising a notch filter positioned between the firstand second microlens array, wherein the notch filter is configured toreject optical energy emitted from the optical waveguide, unwantedscattered optical energy, optical noise, or any combination thereof. 8.The integrated optical system of claim 1, wherein each of the pluralityof sensing regions of the detector comprises a pixel.
 9. The integratedoptical system of claim 8, wherein the pitch of the microlens arraymatches the pitch of the pixels of the detector.
 10. The integratedoptical system of claim 1, wherein each of the plurality of sensingregions receives collected optical energy emitted from a correspondingsingle one of the plurality of nanoholes.
 11. The integrated opticalsystem of claim 10, wherein each of the plurality of sensing regionscomprises a single pixel.
 12. The integrated optical system of claim 1,wherein the optical energy is illumination light.
 13. The integratedoptical system of claim 1, wherein each of the plurality of nanoholescomprises an analyte region.